Gene disruptants producing fatty acyl-CoA derivatives

ABSTRACT

This invention provides microbial organisms, particularly yeasts such as  Yarrowia lipolytica , that have one or more disrupted genes. The gene disruption(s) may yield improved production of fatty acyl-CoA derivatives.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims benefit of priority of U.S. ProvisionalApplication No. 61/427,032, filed Dec. 23, 2010, and of U.S. ProvisionalApplication No. 61/502,697, filed Jun. 29, 2011, the entire content ofeach of which is incorporated herein by reference.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file 90834-820567_ST25.TXT, created onDec. 11, 2011, 188,336 bytes, machine format IBM-PC, MS-Windowsoperating system, is hereby incorporated by reference in its entiretyfor all purposes.

FIELD OF THE INVENTION

This invention relates to modified microbial organisms exhibitingimproved properties, especially improved production of fatty acyl-CoAderivatives.

BACKGROUND OF THE INVENTION

Microbial organisms produce fatty acyl-CoA and fatty acyl-CoAderivatives, such as fatty alcohols, fatty acids, fatty aldehydes, fattyesters, fatty acetates, wax esters, alkanes, and alkenes. Such fattyacyl-CoA derivatives may be used to produce a wide variety of products,including jet and diesel fuels (e.g., biodiesel), chemical surfactants,polymers, nutritional supplements, pharmaceuticals, food additives,cosmetics, and personal care products.

Fatty acids are a principal component of cell membranes and are used byorganisms for energy storage. Fatty acids are metabolized by β-oxidationof fatty acyl-CoA, or conversely, fatty acids are synthesized fromacetyl-CoA by fatty acid synthase multi-enzyme complexes. Fatty alcoholsare the reduction products of fatty acyl-thioester substrates (e.g.,fatty acyl-CoA or fatty acyl-ACP), and like fatty acids, can be producedenzymatically by cultured cells. Enzymes that convert fattyacyl-thioester substrates (e.g., fatty acyl-CoA or fatty acyl-ACP) tofatty alcohols are commonly referred to as “fatty alcohol formingacyl-CoA reductases” or “fatty acyl reductases” (“FARs”).

The commercial production and recovery of fatty alcohols from microbialorganisms is challenging, in part because fatty alcohols are not verystable in many microorganisms. The fatty alcohols (e.g., hexadecanol)can be used as a carbon source for the microorganism, and may thus bemetabolized by the microorganism before recovery for commercialpurposes. The fatty alcohols are likely degraded by enzymes thatcatalyze the oxidation of alkanes to fatty acids (via fatty alcohols).Fatty acids can then be further degraded to acetyl-CoA by enzymes in theβ-oxidation pathway or converted to storage lipids by a set ofacetyltransferases.

Accordingly, there is a need for microbial organisms for the efficientproduction of fatty acyl-CoA derivatives.

BRIEF SUMMARY OF THE INVENTION

This invention provides modified microbial organisms exhibiting improvedproperties, including improved production of fatty acyl-CoA derivatives.In some aspects, the modified microbial organisms have a disrupted genethat confers improved production of fatty acyl-CoA derivatives comparedto a control organism of the same type in which the gene is notdisrupted. In one embodiment the organism is Yarrowia lipolytica.

In one aspect, the invention relates to a microbial organism in whichone or more endogenous genes is disrupted, wherein the endogenous geneis YALI0C17545 or a homolog thereof and/or YALI0E28336 or a homologthereof, and comprising an exogenous gene encoding a functional fattyacyl reductase (FAR) protein operably linked to a promoter. In anotheraspect, both the endogenous YALI0C17545 gene, or homolog thereof, andthe endogenous gene YALI0E28336, or homolog thereof, are disrupted. Inanother aspect, the microbial organism further comprises a disruption ofone or more of endogenous gene YALI0E11099, or a homolog thereof, andendogenous gene YALI0E28534, or a homolog thereof. In another aspect,both the endogenous gene YALI0E11099, or homolog thereof, and theendogenous gene YALI0E28534, or homolog thereof, are disrupted. Inanother aspect, the microbial organism further comprises a disruption ofone or more endogenous genes selected from YALI0B10406, YALI0A19536,YALI0E32769, YALI0E30283, YALI0E12463, YALI0E17787, YALI0B14014,YALI0A10769, YALI0A15147, YALI0A16379, YALI0A20944, YALI0B07755,YALI0B10175, YALI0B13838, YALI0C02387, YALI0C05511, YALI0D01738,YALI0D02167, YALI0D04246, YALI0D05291, YALI0D07986, YALI0D10417,YALI0D14366, YALI0D25630, YALI0E03212, ALI0E07810, YALI0E12859,YALI0E14322, YALI0E15378, YALI0E15400, YALI0E18502, YALI0E18568,YALI0E22781, YALI0E25982, YALI0E28314, YALI0E32417, YALI0F01320,YALI0F06578, YALI0F07535, YALI0F14729, YALI0F22121, YALI0F25003,YALI0E14729, YALI0B17512, and homologs thereof. In another aspect, theendogenous gene YALI0B17512 is disrupted.

In another aspect, two or more of the endogenous genes are disrupted. Inanother aspect, three or more of the endogenous genes are disrupted. Inanother aspect, four or more of the endogenous genes are disrupted.

In another aspect, the microbial organism comprises: a deletion of allor a portion of the coding sequence of the endogenous gene, a mutationin the endogenous gene such that the gene encodes a polypeptide havingreduced activity, antisense RNA or small interfering RNA that inhibitsexpression of the endogenous gene, or a modified regulatory sequencethat reduces expression of the endogenous gene. In one embodiment, themicrobial organism comprises a deletion of all or a portion of thecoding sequence of the endogenous gene.

In one aspect, the exogenous gene encodes a functional FAR proteincomprising a polypeptide sequence having at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to a Marinobacter algicola FAR protein comprising SEQID NO:2. In another aspect, the exogenous gene encodes a functional FARprotein comprising a polypeptide sequence having at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity to a Marinobacter aquaeolei FAR protein comprisingSEQ ID NO:4. In another aspect, the exogenous gene encodes a functionalFAR protein comprising a polypeptide sequence having at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to a Oceanobacter sp. RED65 FAR proteincomprising SEQ ID NO:6. In one aspect, the exogenous gene includes anucleic acid sequence having at least 80% sequence identity, often atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to the nucleic acid sequence of FAR_Maa (SEQID NO:1), FAR_Maq (SEQ ID NO:3), or FAR_Ocs (SEQ ID NO:5). In oneembodiment, the fatty acyl reductase is a gene having at least 80%sequence identity, often at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to thenucleic acid sequence of FAR_Maa (SEQ ID NO:1).

In one aspect, the functional FAR protein is a FAR variant comprisingone or more amino acid substitutions relative to SEQ ID NO:2, 4, or 6,respectively, wherein a cell in which the FAR variant is expressedproduces at least 1.5-fold more fatty acyl-CoA derivatives than acorresponding cell of the same type in which a wild-type FAR proteinfrom which the FAR variant is derived is expressed. In another aspect,the exogenous FAR gene encodes a FAR variant that comprises from 1 toabout 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, or 40 amino acid substitutions relative toFAR_Maa (SEQ ID NO:2), FAR_Maq (SEQ ID NO:4), or FAR_Ocs (SEQ ID NO:6).In one embodiment, the exogenous FAR gene encodes a FAR variant thatcomprises from 1 to about 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or 40 amino acidsubstitutions relative to FAR_Maa (SEQ ID NO:2).

In another aspect, the microbial organism has multiple copies of theendogenous gene (e.g., a diploid number) and more than one copy of theendogenous gene is disrupted. In another aspect, the microbial organismexpresses multiple copies of the exogenous gene. In another aspect, theexogenous gene is integrated into the genome of the microbial organism.

In another aspect, the microbial organism further comprises a secondexogenous gene that encodes a fatty acid synthase (FAS), an estersynthase, an acyl-ACP thioesterase (TE), a fatty acyl-CoA synthase(FACS), an acetyl-CoA carboxylase (ACC), a xylose isomerase, or aninvertase.

In one aspect, the microbial organism is algae, bacteria, mold,filamentous fungus, or yeast, such as an oleaginous yeast. In oneaspect, the microbial organism is a yeast. In one aspect, the yeast isYarrowia, Brettanomyces, Candida, Cryptococcus, Endomycopsis, Hansenula,Kluyveromyces, Lipomyces, Pachysolen, Pichia, Rhodosporidium,Rhodotorula, Saccharomyces, Schizosaccharomyces, Trichosporon, orTrigonopsis. In one aspect, the yeast is an oleaginous yeast, such asYarrowia lipolytica, Yarrowia paralipolytica, Candida revkauji, Candidapulcherrima, Candida tropicalis, Candida utilis, Candida curvata D,Candida curvataR, Candida diddensiae, Candida boldinii, Rhodotorulaglutinous, Rhodotorula graminis, Rhodotorula mucilaginosa, Rhodotorulaminuta, Rhodotorula bacarum, Rhodosporidium toruloides, Cryptococcus(terricolus) albidus var. albidus, Cryptococcus laurentii, Trichosporonpullans, Trichosporon cutaneum, Trichosporon cutancum, Trichosporonpullulans, Lipomyces starkeyii, Lipomyces lipoferus, Lipomycestetrasporus, Endomycopsis vernalis, Hansenula ciferri, Hansenulasaturnus, or Trigonopsis variabilis. In one aspect, the yeast isYarrowia lipolytica.

In another aspect, the microbial organism exhibits at least a 1-fold, atleast a 1.2-fold, at least a 1.5-fold, at least a 4-fold, or at least a20-fold increase in the production of a fatty acyl-CoA derivativecompared to a control organism of the same type (e.g., an otherwiseidentical control microbial organism in which the one or more genes arenot disrupted).

In another aspect, the invention relates to a microbial organismcomprising one or more disrupted endogenous genes, wherein at least oneof the disrupted genes is YALI0C17545, YALI0E28336, YALI0E11099,YALI0B10406, YALI0A19536, YALI0E28534, YALI0E32769, YALI0E30283,YALI0E12463, YALI0E17787, YALI0B14014, YALI0A10769, YALI0A15147,YALI0A16379, YALI0A20944, YALI0B07755, YALI0B10175, YALI0B13838,YALI0C02387, YALI0C05511, YALI0D01738, YALI0D02167, YALI0D04246,YALI0D05291, YALI0D07986, YALI0D10417, YALI0D14366, YALI0D25630,YALI0E03212, ALI0E07810, YALI0E12859, YALI0E14322, YALI0E15378,YALI0E15400, YALI0E18502, YALI0E18568, YALI0E22781, YALI0E25982,YALI0E28314, YALI0E32417, YALI0F01320, YALI0F06578, YALI0F07535,YALI0F14729, YALI0F22121, YALI0F25003, YALI0E14729, YALI0B17512, or ahomolog of any of these, and an exogenous gene encoding a functionalfatty acyl reductase operably linked to a promoter, wherein themicrobial organism exhibits at least a 1-fold, at least a 1.2-fold, atleast a 1.5-fold, at least a 4-fold, or at least a 20-fold increase inthe production of a fatty acyl-CoA derivative compared to a controlorganism of the same type (e.g., an otherwise identical controlmicrobial organism in which the one or more genes are not disrupted).

In yet another aspect, at least one of the disrupted endogenous genes isYALI0C17545, YALI0E28336, YALI0E11099, YALI0B10406, YALI0A19536,YALI0E28534, YALI0E32769, YALI0E30283, YALI0E12463, YALI0E14729,YALI0B17512, or a homolog of any of these.

In one aspect, YALI0C17545 or a homolog thereof is disrupted. In anotheraspect, YALI0E28336 or a homolog thereof is disrupted. In yet anotheraspect, both YALI0C17545 or a homolog thereof and YALI0E28336 or ahomolog thereof are disrupted.

In yet another aspect, the microbial organism further comprises a seconddisrupted gene that is YALI0C17545, YALI0E28336, YALI0E11099,YALI0B10406, YALI0A19536, YALI0E28534, YALI0E32769, YALI0E30283,YALI0E12463, YALI0E17787, YALI0B14014, YALI0A10769, YALI0A15147,YALI0A16379, YALI0A20944, YALI0B07755, YALI0B10175, YALI0B13838,YALI0C02387, YALI0D05511, YALI0D01738, YALI0D02167, YALI0D04246,YALI0D05291, YALI0D07986, YALI0D10417, YALI0D14366, YALI0D25630,YALI0E03212, ALI0E07810, YALI0E12859, YALI0E14322, YALI0E15378,YALI0E15400, YALI0E18502, YALI0E18568, YALI0E22781, YALI0E25982,YALI0E28314, YALI0E32417, YALI0F01320, YALI0F06578, YALI0F07535,YALI0F14729, YALI0F22121, YALI0F25003, YALI0E14729, YALI0B17512 or ahomolog of any of these.

In one aspect, the microbial organism comprises two disrupted endogenousgenes. When two genes are disrupted, YALI0C17545 or a homolog thereofand/or YALI0E30283 or a homolog thereof can be disrupted. In anotheraspect, the microbial organism comprises three disrupted endogenousgenes. In yet another aspect, the microbial organism comprises four ormore disrupted endogenous genes.

In another aspect, the microbial organism comprises a combination ofdisrupted endogenous genes, or homologs thereof. The combination can be:

a. YALI0C17545 and YALI0E28336;

b. YALI0C17545 and YALI0B10406;

c. YALI0C17545 and YALI0E28534;

d. YALI0C17545 and YALI0E30283;

e. YALI0E28336 and YALI0E30283;

f. YALI0E11099 and YALI0E30283;

g. YALI0A19536 and YALI0E30283;

h. YALI0A19536 and YALI0E28534;

i. YALI0E30283 and YALI0E12463;

j. YALI0B10406 and YALI0E14729;

k. YALI0C17545 and YALI0E14729;

l. YALI0E11099 and YALI0E14729;

m. YALI0C17545, YALI0E28336, and YALI0E11099;

n. YALI0C17545, YALI0E28336, and YALI0B10406;

o. YALI0C17545, YALI0E28336, and YALI0A19536;

p. YALI0C17545, YALI0E28336, and YALI0E28534;

q. YALI0C17545, YALI0E28336, and YALI0E32769;

r. YALI0C17545, YALI0E28336, and YALI0E12463;

s. YALI0C17545, YALI0E11099, and YALI0B10406;

t. YALI0C17545, YALI0B10406, and YALI0A19536;

u. YALI0E28336, YALI0E11099, and YALI0B10406;

v. YALI0E11099, YALI0B10406, and YALI0A19536;

w. YALI0C17545, YALI0E28534, and YALI0B17512;

x. YALI0E11099, YALI0A19536, YALI0B10406, and YALI0B17512;

y. YALI0C17545, YALI0E28336, YALI0E11099, and YALI0B10406;

z. YALI0C17545, YALI0E28336, YALI0E11099, and YALI0A19536;

aa. YALI0C17545, YALI0E28336, YALI0E11099, and YALI0E28534;

bb. YALI0C17545, YALI0E28336, YALI0E11099, and YALI0E32769;

cc. YALI0C17545, YALI0E28336, YALI0B10406, and YALI0A19536;

dd. YALI0C17545, YALI0E28336, YALI0B10406, and YALI0E32769;

ee. YALI0C17545, YALI0E28336, YALI0A19536, and YALI0E28534;

ff. YALI0C17545, YALI0E28336, YALI0E28534, and YALI0E32769;

gg. YALI0C17545, YALI0E28336, YALI0E28534, and YALI0E12463;

hh. YALI0E28336, YALI0E11099, YALI0B10406, and YALI0E32769; or

ii. YALI0E11099, YALI0E28336, YALI0C17545, and YALI0E14729.

In one aspect, a Yarrowia lipolytica cell comprises one or moredisrupted endogenous genes, wherein at least one disrupted gene isYALI0C17545, YALI0E28336, YALI0E11099, YALI0B10406, YALI0A19536,YALI0E28534, YALI0E32769, YALI0E30283, YALI0E12463, YALI0E14720,YALI0B17512, or a homolog of any of these, and an exogenous geneencoding a functional fatty acyl reductase operably linked to apromoter, wherein the Yarrowia lipolytica cell exhibits at least a1-fold, at least a 1.2-fold, at least a 1.5-fold, at least a 4-fold, orat least a 20-fold increase in the production of a fatty acyl-CoAderivative compared to a control organism of the same type (e.g., anotherwise identical control microbial organism in which the one or moregenes are not disrupted). In one aspect, the exogenous gene includes anucleic acid sequence having at least 80% sequence identity, often atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to a nucleic acid sequenceof FAR_Maa (SEQ ID NO:1), FAR_Maq (SEQ ID NO:3), or FAR_Ocs (SEQ IDNO:5), or it encodes a polypeptide that includes an amino acid sequencehaving at least 80% sequence identity, often at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to a polypeptide of FAR_Maa (SEQ ID NO:2), FAR_Maq(SEQ ID NO:4), or FAR_Ocs (SEQ ID NO:6); or encodes a FAR variantpolypeptide that comprises from 1 to about 50, e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or 40 aminoacid substitutions relative to FAR_Maa (SEQ ID NO:2), FAR_Maq (SEQ IDNO:4), or FAR_Ocs (SEQ ID NO:6). In one embodiment, the exogenous FARgene encodes a FAR variant that comprises from 1 to about 50, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, or 40 amino acid substitutions relative to FAR_Maa (SEQ ID NO:2).

In another aspect, the invention provides a microbial organism in whichone or more endogenous genes is disrupted, wherein the endogenous geneis selected from YALI0C17545, YALI0E28336, YALI0E11099, YALI0B10406,YALI0A19536, YALI0E28534, YALI0E32769, YALI0E30283, YALI0E12463,YALI0E17787, YALI0B14014, YALI0A10769, YALI0A15147, YALI0A16379,YALI0A20944, YALI0B07755, YALI0B10175, YALI0B13838, YALI0D02387,YALI0D05511, YALI0D01738, YALI0D02167, YALI0D04246, YALI0D05291,YALI0D07986, YALI0D10417, YALI0D14366, YALI0D25630, YALI0E03212,ALI0E07810, YALI0E12859, YALI0E14322, YALI0E15378, YALI0E15400,YALI0E18502, YALI0E18568, YALI0E22781, YALI0E25982, YALI0E28314,YALI0E32417, YALI0F01320, YALI0F06578, YALI0F07535, YALI0F14729,YALI0F22121, YALI0F25003, YALI0E14729, YALI0B17512, and homologsthereof. In another aspect, the endogenous gene YALI0B17512, or homologthereof, is disrupted. In another aspect, YALI0B17512 encodes apolypeptide comprising a cytoplasmic domain and the disruption comprisesa deletion of at least a portion of the cytoplasmic domain.

In another aspect, one or more of the endogenous gene YALI0C17545, orhomolog thereof, and the endogenous gene YALI0E28336, or homologthereof, is disrupted.

In another aspect, the invention provides a method for producing a fattyacyl-CoA derivative comprising providing a microbial organism asdescribed herein; and culturing the microbial organism under conditionsin which fatty acyl-CoA derivatives are produced. The method can furtherinclude recovering (e.g., isolating) the fatty acyl-CoA derivative. Inone aspect, at least 5 g/L or at least 15 g/L of fatty acyl-CoAderivatives per liter of culture medium is produced.

In another aspect, a method for producing a fatty acyl-CoA derivativecan include contacting a cellulose-containing biomass with one or morecellulases to yield fermentable sugars; and contacting the fermentablesugars with the microbial organism. In another aspect, the method forproducing a fatty acyl-CoA derivative can include contacting fermentablesugars comprising sucrose with the microorganism as described herein.

In one aspect, the fatty acyl-CoA derivative is a fatty alcohol, fattyacid, fatty aldehyde, fatty ester, fatty acetate, wax ester, alkane, oralkene. In another aspect, the fatty acyl-CoA derivative is a fattyalcohol. In one aspect, the fatty acyl-CoA derivative has a carbon chainlength of 8 to 24 carbon atoms, such as a fatty alcohol with 8 to 24carbon atoms.

In another aspect, the invention provides a composition comprising thefatty acyl-CoA derivative(s) produced by a method as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates routes to biosynthesis of fatty acyl-CoA derivativesin Y. lipolytica. Native pathways for biosynthesis of fatty acyl-CoAfrom glucose (reactions 1-3) and for degradation of alkanes and productsof alkane oxidation to fatty acyl-CoA are shown (reactions 4-7). Nativeand exogenous pathways for production of fatty acyl-CoA derived productsare also shown, and include: acyltransferases (triacylglycerides),thioesterases (fatty acids), ester synthases (esters), acyl-CoAreductases (“FARs”) (fatty aldehydes and fatty alcohols), and aldehydedecarbonylases (alkanes).

FIG. 2 illustrates plasmid pCEN411 for expression of FAR genes in Y.lipolytica.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in analyticalchemistry, cell culture, molecular genetics, organic chemistry, andnucleic acid chemistry and hybridization described below are those wellknown and commonly employed in the art. It is noted that as used herein,“a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. The term “comprising” and its cognates areused in their inclusive sense; that is, equivalent to the term“including” and its corresponding cognates.

The techniques and procedures are generally performed according toconventional methods in the art and various general references. See,e.g., Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rded.; Ausubel, ed., 1990-2008, Current Protocols in Molecular Biology.Standard techniques, or modifications thereof, are used for nucleic acidand polypeptide synthesis and for chemical syntheses and chemicalanalyses. Generally, enzymatic reactions and purification steps areperformed according to the manufacturer's specifications. For techniquesregarding yeast recombinant techniques, nutrition, and growth, see,e.g., Walker, 1998, Yeast Physiology and Biotechnology.

The term “disrupted,” as applied to a gene, refers to any geneticmodification that decreases or eliminates the expression of the geneand/or the functional activity of the corresponding gene product (mRNAand/or protein). Genetic modifications include complete or partialinactivation, suppression, deletion, interruption, blockage, ordown-regulation of a gene. This can be accomplished, for example, bygene “knockout,” inactivation, mutation (e.g., insertion, deletion,point, or frameshift mutations that disrupt the expression or activityof the gene product), or by use of inhibitory RNAs (e.g., sense,antisense, or RNAi technology). A disruption may encompass all or partof a gene's coding sequence.

The term “knockout” has its conventional meaning in the art, and refersto an organism or cell in which a specific gene has been inactivated bygenetic manipulation, generally by a recombination event in which all ora portion of gene is deleted or a heterologous DNA is inserted, so thatthe cell or organism does not produce a functional product encoded bythe gene. Knockout also refers to the process of making an organism orcell with an inactivated gene, usually by replacing at least a portionof a coding sequence of a gene with an artificial piece of DNA (e.g.,encoding a selection marker) and/or deleting at least a portion of thecoding sequence of the gene, so that a functional gene product is notexpressed in the cell or organism. In some embodiments the entire codingsequence of the gene is excised.

“Coding sequence” refers to that portion of a nucleic acid that encodesfor an amino acid sequence of a protein.

The term “expression” includes any step involved in the production of apolypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

The term “fatty acyl-CoA derivative” is a compound that can bemetabolically derived from fatty acyl-CoA, fatty acyl-ACP, or othersimilar fatty acyl thioester in a microorganism. Derivatives include,but are not limited to, fatty alcohols, fatty acids, fatty aldehydes,fatty esters, fatty acetates, wax esters, alkanes, and alkenes.Saturated or unsaturated fatty acyl-CoA derivatives can be describedusing the notation “Ca:b,” where “a” is an integer that represents thetotal number of carbon atoms, and “b” is an integer that refers to thenumber of double bonds in carbon chain. Unsaturated fatty acyl Co-Aderivatives can be referred to as “cisΔ^(x)” or “transΔ^(x)” wherein“cis” and “trans” refer to the carbon chain configuration around thedouble bond. The “x” indicates the number of the first carbon of thedouble bond, where carbon 1 is, e.g., the carboxylic acid carbon of thefatty acid or the carbon bound to the —OH group of the fatty alcohol.For the derivatives described below, “R” is a C₈ to C₂₄ saturated,unsaturated, linear, branched, or cyclic hydrocarbon (or “C₇ to C₂₃” inderivative formulas expressly articulating the terminal carbon).

The term “fatty alcohol” as used herein refers to an aliphatic alcoholof the formula R—OH, where “R” is as defined above. In some embodiments,a fatty alcohol produced according to the methods disclosed herein is aC8-C24 saturated or unsaturated fatty alcohol (i.e., a C8, C9, C10, C11,C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, or C24 fattyalcohol). In some embodiments, one or more of the following fattyalcohols is produced: 1-octanol (C8:0), 1-decanol (C10:0), 1-dodecanol(C12:0), 1-tetradecanol (C14:0), 1-hexadecanol (C16:0), 1-octadecanol(C18:0), 1-icosanol (C20:0), 1-docosanol (C22:0), 1-tetracosanol(C24:0), cis Δ⁹-1-hexadecenol (C16:1), and cis Δ¹¹-1-octadecenol(C18:1). It is understood that, unless otherwise specified, a referenceto a “Cx fatty alcohol” includes both saturated and unsaturated fattyalcohols having “x” carbon atoms.

The term “fatty acid” as used herein refers to a compound of the formula

The term “fatty aldehyde” as used herein refers to a compound of theformula

The term “fatty esters” includes compounds of the formula

where R′ is a short chain, e.g., C1 to C6, preferably C1 to C4hydrocarbon. For example, fatty acyl-CoA can be reacted with a shortchain alcohol (e.g., methanol or ethanol) to form conventional fattyesters. Conversely, fatty alcohols can be reacted with short chainthioesters (e.g., acetyl CoA) to form esters. Both ester types areencompassed by the term “fatty esters.”

The term “fatty acetates” as used herein refers to a compound of theformula

The term “wax esters” as used herein refers to an ester derived from along chain fatty acid and a long chain alcohol.

Reference herein to particular endogenous genes by name is forillustration and not limitation. It is understood that gene names varyfrom organism to organism and reference to a gene name is not intendedto be limiting, but is intended to encompass homologs (i.e., which maybe endogenous to a related microbial organism) and polymorphic variants.Homologs and variants can be identified based on sequence identityand/or similar biological (e.g., enzymatic) activity. In certainembodiments, the invention includes a polynucleotide or polypeptidesequence with at least 50%, 60%, 70%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the named gene or gene product.

“Identity” or “percent identity,” in the context of two or morepolynucleotide or polypeptide sequences, refers to two or more sequencesor sub-sequences that are the same or have a specified percentage ofnucleotides or amino acid residues, respectively, that are the same.Percent identity may be determined by comparing two optimally alignedsequences over a comparison window, wherein the portion of thepolynucleotide or polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which may also contain gaps to optimize thealignment) for alignment of the two sequences. For example, the sequencecan have a percent identity of at least 50%, 60%, 70%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a specified region to areference sequence when compared and aligned for maximum correspondenceover a comparison window, or designated region as measured using asequence comparison algorithms or by manual alignment and visualinspection.

Alignment of sequences for comparison can be conducted, e.g., by thelocal homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math.2:482, by the homology alignment algorithm of Needleman and Wunsch,1970, J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the GCG Wisconsin Software Package), or by visualinspection (see generally, Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, John Wiley & Sons, Inc. (1995Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., 1990, J. Mol. Biol.215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Informationwebsite. This algorithm involves first identifying high scoring sequencepairs (HSPs) by identifying short words of length W in the querysequence, which either match or satisfy some positive-valued thresholdscore T when aligned with a word of the same length in a databasesequence. T is referred to as, the neighborhood word score threshold(Altschul et al, supra). These initial neighborhood word hits act asseeds for initiating searches to find longer HSPs containing them. Theword hits are then extended in both directions along each sequence foras far as the cumulative alignment score can be increased. Cumulativescores are calculated using, for nucleotide sequences, the parameters M(reward score for a pair of matching residues; always >0) and N (penaltyscore for mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (VV) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA89:10915). Exemplary determination of sequence alignment and % sequenceidentity can employ the BESTFIT or GAP programs in the GCG WisconsinSoftware package (Accelrys, Madison Wis.), using default parametersprovided.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, at least 100 residues in length or the fulllength of the nucleic acid or polypeptide. Since two polynucleotides orpolypeptides may each (1) comprise a sequence (i.e., a portion of thecomplete sequence) that is similar between the two sequences, and (2)may further comprise a sequence that is divergent between the twosequences, sequence comparisons between two (or more) polynucleotides orpolypeptide are typically performed by comparing sequences of the twopolynucleotides over a “comparison window” to identify and compare localregions of sequence similarity.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

As used herein, “polynucleotide” refers to a polymer ofdeoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form, and complements thereof.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of amino acid residues.

“Improved production” refers to an increase in the amount of measurablefatty acyl-CoA derivatives produced by a modified microbial organism(i.e., a microbial organism in which one or more endogenous genes isdisrupted) as compared to the amount produced by a control microbialorganism of the same type in which the genes are not disrupted, whencultured under the same conditions. “Control organism of the same type”means an organism of the same species having a genome that isessentially identical to the genome of the modified microbial organism,except for a disrupted gene or combination of genes described here. Forexample, a Y. lipolytica strain (e.g., DSMZ 1345) in which a fatty acidsynthase is overexpressed would be a “control organism of the same type”for the same Y. lipolytica strain (e.g., DSMZ 1345) in which a fattyacid synthase is overexpressed and in which the specified gene orcombination of genes is disrupted. The term “otherwise identicalorganism” is used interchangeably with “control organism of the sametype.” The improved production may occur by any mechanism, e.g.,increased production and/or decreased degradation or utilization.

The term “functional,” as used in reference to a polypeptide, means thatthe polypeptide exhibits catalytic activity in vivo. The term“functional” can be used interchangeably with the term “biologicallyactive.”

The terms “wild-type” or “native” used in reference to a polypeptide orprotein mean a polypeptide or protein expressed by a microorganism foundin nature. When used in reference to a microorganism, the term means anaturally occurring (not genetically modified) microorganism.

A “FAR” (also known as “fatty alcohol forming acyl-CoA reductase” or“fatty acyl reductase”) as used herein refers to an enzyme that convertsfatty acyl-thioester substrates (e.g., fatty acyl-CoA or fatty acyl-ACP)to fatty alcohols. “CoA” is a non-protein acyl carrier group factor (ormoiety) involved in the synthesis and oxidation of fatty acids. “ACP” isa polypeptide or protein subunit of fatty acid synthase used in thesynthesis of fatty acids.

The term “wild-type FAR,” as used herein, refers to a FAR polypeptidethat is produced in nature. In some embodiments, a wild-type FAR isproduced by a gammaproteobacteria, including but not limited to strainsof Marinobacter, Oceanobacter, and Hahella. Naturally occurring FARpolypeptides are described, for example, in US patent publication2011/0000125, incorporated by reference herein. In some embodiments, awild-type FAR is a naturally-occurring FAR polypeptide that is producedby the Marinobacter algicola strain DG893 (SEQ ID NO:2). In someembodiments, a wild-type FAR is a naturally-occurring FAR polypeptidethat is produced by the Marinobacter aquaeolei strain VT8 (SEQ ID NO:4)In some embodiments, a wild-type FAR is a naturally-occurring FARpolypeptide that is produced by Oceanobacter sp. RED65 (SEQ ID NO:6).

The term “FAR variant,” as used herein, refers to full-length FARpolypeptides having substitutions at one or more amino acid positionsrelative to a wild-type FAR polypeptide, and functional fragmentsthereof, wherein a cell (e.g., a microbe) in which the variant isexpressed is capable of catalyzing increased production of fattyalcohols as compared to a cell in which the wild-type FAR polypeptide isexpressed. In some embodiments, a FAR variant comprises at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to a FARpolypeptide of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 and alsocomprises one or more amino acid substitutions that give rise toincreased fatty acyl-CoA derivative (e.g., fatty alcohol) production ascompared to the fatty acyl-CoA derivative production that can beachieved with the wild-type FAR polypeptide from which it is derived.FAR variants are described, for example, in U.S. application Ser. No.13/171,138, incorporated by reference herein. As used herein, exceptwhere otherwise clear from context, reference to a “FAR,” “FAR protein,”“FAR variant,” or “FAR fragment” is intended to refer to a functionalFAR protein, functional FAR variant, or functional FAR fragment, even ifnot explicitly indicated.

The term “endogenous” refers to a gene or protein that is originallycontained within an organism (i.e., encodes a sequence found in thewild-type organism). Conversely, the terms “exogenous” or“heterologous,” as used in reference to a gene, refer interchangeably toa gene that originates outside the microorganism, such as a gene fromanother species, or a modified or recombinant gene. An exogenous orheterologous gene may be introduced into the microorganism by methodsknown in the art.

Nucleic acid sequences may be “introduced” into a cell by transfection,transduction, transformation, or any other method. A nucleic acidsequence introduced into a eukaryotic or prokaryotic cell may beintegrated into a chromosome or may be maintained in an episome.

The terms “transform” or “transformation,” as used in reference to acell, means a cell has a non-native nucleic acid sequence integratedinto its genome or as an episome (e.g., plasmid) that is maintainedthrough multiple generations.

“Vector” refers to a DNA construct comprising a DNA protein codingsequence. A vector may be an expression vector comprising a proteincoding sequence operably linked to a suitable control sequence (i.e.,promoter) capable of effecting the expression of the DNA in a suitablehost.

“Operably linked” means that DNA sequence segments are arranged so thatthey function in concert for their intended purposes, e.g., a promotercontrols transcription of a gene sequence to which it is operablylinked.

“Promoter sequence” is a nucleic acid sequence that is recognized by acell for expression of the coding region. The control sequence maycomprise an appropriate promoter sequence. The promoter sequencecontains transcriptional control sequences, which mediate the expressionof the polypeptide. The promoter may be any nucleic acid sequence whichshows transcriptional activity in the cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either endogenous orexogenous (heterologous) to the host cell.

The term “culturing” refers to growing a population of microbial cellsunder suitable conditions in a liquid or solid medium. Most often aliquid medium is used. In some embodiments, culturing refers to thefermentative bioconversion of a substrate to an end product.

The term “contacting” refers to combining an enzyme and a substrateunder conditions in which the enzyme can act on the substrate. Thoseskilled in the art will recognize that mixing a solution containing anenzyme (e.g., a cellulase) with a substrate (e.g., acellulose-containing biomass) will effect “contacting.” Similarly, inthe context of culturing microorganisms, culturing microorganisms in amedium containing a substrate (e.g., a fermentable sugar) will effect“contacting” the microorganism with the substrate.

The term “cellulase” refers to a category of enzymes capable ofdisrupting the crystalline structure of cellulose and hydrolyzingcellulose (β-1,4-glucan or β-D-glucosidic linkages) to shorteroligosaccharides, disaccharides (e.g., cellobiose), and/ormonosaccharides (e.g., glucose). Cellulases include endoglucanases,cellobiohydrolases, and beta-glucosidases.

The terms “cellulose-containing biomass,” “cellulosic biomass,” and“cellulosic substrate” refer to materials that include cellulose.Biomass can be derived from plants, animals, or microorganisms, and mayinclude agricultural, industrial, and forestry residues, municipal solidwastes, industrial wastes, and terrestrial and aquatic crops grown forenergy purposes. Examples of biomass include, but are not limited to,wood, wood pulp, paper pulp, corn fiber, corn grain, corn cobs, cropresidues such as corn husks, corn stover, grasses, wheat, wheat straw,barley, barley straw, hay, rice straw, switchgrass, waste paper, paperand pulp processing waste, woody or herbaceous plants, fruit orvegetable pulp, distillers grain, rice hulls, cotton, hemp, flax, sisal,sugar cane bagasse, sorghum, soy, components obtained from milling ofgrains, trees, branches, roots, leaves, wood chips, sawdust, shrubs andbushes, vegetables, fruits, flowers, animal manure, and mixturesthereof.

“Fermentable sugar” means simple sugars (monosaccharides, disaccharides,and short oligosaccharides) including but not limited to glucose,fructose, xylose, galactose, arabinose, mannose, and sucrose.

The term “recoverable fatty acyl-CoA derivative” refers to the amount offatty acyl-CoA derivatives that can be isolated from a reaction mixtureyielding the fatty acyl-CoA derivatives according to methods known inthe art.

II. Introduction

We have discovered that, surprisingly, disruption of certain endogenousgenes and combinations of genes in a microbial organism, e.g., Yarrowialipolytica, expressing a fatty acyl reductase (FAR) results in increasedproduction of fatty acyl-CoA derivatives. A FAR (also known as “fattyalcohol forming acyl-CoA reductase” or “fatty acyl reductase”) refers toan enzyme that catalyzes the reduction of a fatty acyl-CoA, a fattyacyl-ACP, or other fatty acyl thioester complex to a fatty alcohol, in areaction linked to the oxidation of NAD(P)H to NAD(P)⁺, as shown in thefollowing Scheme 1:

wherein “R” represents a C7 to C23 saturated, unsaturated, linear,branched or cyclic hydrocarbon chain, and “R₁” represents CoA, ACP orother fatty acyl thioester substrates. CoA is a non-protein acyl carriergroup factor (or moiety) involved in the synthesis and oxidation offatty acids. “ACP” is a polypeptide or protein subunit of fatty acidsynthase used in the synthesis of fatty acids. In some embodiments, afatty aldehyde intermediate may be produced in the reaction depicted inScheme 1.

Wild-type FAR proteins have been described in WO 2011/008535 (published20 Jan. 2011), incorporated by reference herein for all purposes.Certain FAR enzymes isolated from genera of the class of marine bacteriasuch as gammaproteobacteria found in seawater (and particularly FARsobtained from strains of Marinobacter and Oceanobacter or taxonomicequivalents thereof) are capable of generating high yields of fattyalcohols when genes encoding these enzymes are expressed in heterologouscells. As described in the Examples section below, it has now beendiscovered that microbial organisms in which certain genes orcombinations of genes are disrupted and which express a gene encoding aFAR protein have increased production of fatty acyl-CoA derivatives, ascompared to otherwise identical microbial organisms expressing theexogenous gene encoding the FAR protein in which genes have not beendisrupted. Thus, in one aspect the present invention relates to amicrobial organism exhibiting increased production of fatty acyl-CoAderivatives, wherein the microbial organism comprises one or moredisrupted endogenous genes and an exogenous gene encoding a FAR protein.These modified microbial organisms may be used in commercial productionof fatty acyl-CoA derivatives.

Various aspects of the invention are described in the followingsections.

III. Disruption of Endogenous Genes

Endogenous Genes for Disruption

In one aspect, the present invention relates to recombinant microbialorganisms, such as yeasts, in which one or more endogenous genes aredisrupted, and which exhibit improved production of fatty acyl-CoAderivatives, and methods of using such microbial organisms.

The endogenous genes described herein are named with reference to theYarrowia lipolytica genome. Dujon, et al., 2004, “Genome evolution inyeasts” Nature 430:35-44. The abbreviated gene name (e.g., “C17545”) andthe full gene name (e.g., “YALI0C17545”) are used interchangeably, andboth encompass polymorphic variants of the gene. In some embodiments,the host cell is other than Y. lipolytica, and the endogenous gene is ahomolog of the Y. lipolytica gene. As noted above, gene names vary fromorganism to organism and any gene name used herein is not intended to belimiting, but is intended to encompass homologs as well. Table 1provides a listing of nucleotide sequences for exemplary disrupted genesfrom Y. lipolytica as well as activities of the encoded proteins.Biological activities are assigned based on reference to the scientificliterature and/or based on functional and sequence characterization.While the known or predicted biological activities may be useful foridentifying homologs, a nucleotide sequence and/or protein for use inthe present invention is not limited to those nucleotide sequencesand/or proteins that have previously been identified as being involvedin fatty acyl-CoA derivative production.

In some embodiments, a microbial organism of the present invention(e.g., algae, bacteria, mold, filamentous fungus, or yeast, e.g.,Yarrowia lipolytica) has one or more disrupted endogenous genes selectedfrom the genes listed in Table 1 and homologs thereof.

TABLE 1 Nucleotide sequences for disrupted genes in Yarrowia lipolyticaY. lipolytica SEQ ID NO. gene name (DNA) Known or Predicted BiologicalActivity YALI0C17545 7 Phosphatidylinositol transfer protein YALI0E283368 YALI0E11099 9 Beta-oxidation enzyme PAT1 YALI0B10406 10 Enoyl-CoAhydratase YALI0A19536 11 Alcohol dehydrogenase YALI0E28534 12YALI0E32769 13 Acyltransferase DGAT2 YALI0E30283 14 GUP1 YALI0E12463 15SOR1 YALI0E17787 16 Fatty alcohol dehydrogenase ADH2 YALI0B14014 17 GMCoxidoreductase YALI0A10769 18 Alcohol dehydrogenase YALI0A15147 19 Fattyalcohol dehydrogenase ADH4 YALI0A16379 20 Fatty alcohol dehydrogenaseADH3 YALI0A20944 21 Peroxisomal membrane protein YALI0B07755 22 CoAligase YALI0B10175 23 Alcohol dehydrogenase YALI0B13838 24 Alkanemonooxygenase ALK5 YALI0C02387 25 Transcription factor YAS1 YALI0C0551126 Phosphatidylinositol transfer protein YALI0D01738 27 Alcoholdehydrogenase YALI0D02167 28 Alcohol dehydrogenase YALI0D04246 29Peroxisomal membrane protein PXA2 YALI0D05291 30 Transcription factorSCS2 YALI0D07986 31 Acyltransferase DGAT1 YALI0D10417 32 YALI0D14366 33YALI0D25630 34 Fatty alcohol dehydrogenase ADH1 YALI0E03212 35 FADbinding oxidoreductase YALI0E07810 36 Alcohol dehydrogenase YALI0E1285937 Acyl-CoA ligase YALI0E14322 38 2,4-dienoyl-CoA reductase YALI0E1537839 Beta-oxidation enzyme MFE2 YALI0E15400 40 Fatty aldehydedehydrogenase ALDH2 YALI0E18502 41 Flavoprotein oxygenase YALI0E18568 42Beta-oxidation enzyme POT1 YALI0E22781 43 Oxysterol binding proteinYALI0E25982 44 Alkane monooxygenase ALK1 YALI0E28314 45 YALI0E32417 46Transcription factor YAS2 YALI0F01320 47 Alkane monooxygenase ALK2YALI0F06578 48 Acyltransferase ARE2 YALI0F07535 49 YALI0F14729 50Thioesterase YALI0F22121 51 Enoyl-CoA hydratase YALI0F25003 52 Alcoholdehydrogenase YALI0E14729g 53 ABC1 alkane transporter YALI0B17512g 54Sec62 ER protein translocase

In some embodiments, the microbial organism, e.g. Yarrowia lipolytica,has one or more endogenous genes disrupted, wherein at least one of thedisrupted genes is YALI0C17545, YALI0E28336, YALI0E11099, YALI0B10406,YALI0A19536, YALI0E28534, YALI0E32769, YALI0E30283, YALI0E12463,YALI0E17787, YALI0B14014, YALI0A10769, YALI0A15147, YALI0A16379,YALI0A20944, YALI0B07755, YALI0B10175, YALI0B13838, YALI0C02387,YALI0D05511, YALI0D01738, YALI0D02167, YALI0D04246, YALI0D05291,YALI0D07986, YALI0D10417, YALI0D14366, YALI0D25630, YALI0E03212,ALI0E07810, YALI0E12859, YALI0E14322, YALI0E15378, YALI0E15400,YALI0E18502, YALI0E18568, YALI0E22781, YALI0E25982, YALI0E28314,YALI0E32417, YALI0F01320, YALI0F06578, YALI0F07535, YALI0F14729,YALI0F22121, YALI0F25003, YALI0E14729, YALI0B17512, or a homolog of anyof these.

In some embodiments, the disrupted endogenous gene is C17545 (SEQ IDNO:7) or a homolog thereof. In some embodiments, the disruptedendogenous gene is E28336 (SEQ ID NO:8) or a homolog thereof. In someembodiments, the disrupted endogenous gene is E11099 (SEQ ID NO:9) or ahomolog thereof. In some embodiments, the disrupted endogenous gene isE28534 (SEQ ID NO:12) or a homolog thereof. In some embodiments, thedisrupted endogenous gene is 817512 (SEQ ID NO:54) or a homolog thereof.

In some embodiments, the microbial organism, e.g. Yarrowia lipolytica,is in which one, two, three, four, or five endogenous genes in themicrobial organism are disrupted. In some embodiments, one or more, twoor more, three or more, four or more, or five or more endogenous genesare disrupted. Microbial organisms with multiple disrupted endogenousgenes may advantageously exhibit synergistic effects, as has beenobserved in yeast (see Examples, below). The present invention includesbut is not limited to exemplary embodiments shown in the Examplessection. In some embodiments, the microbial organism has two, three, orfour disrupted endogenous genes.

In another embodiment, the microbial organism has at least two disruptedendogenous genes. In some embodiments, both the first disrupted gene andthe second disrupted gene are selected from the following: YALI0C17545,YALI0E28336, YALI0E11099, YALI0B10406, YALI0A19536, YALI0E28534,YALI0E32769, YALI0E30283, YALI0E12463, YALI0E17787, YALI0B14014,YALI0A10769, YALI0A15147, YALI0A16379, YALI0A20944, YALI0B07755,YALI0B10175, YALI0B13838, YALI0C02387, YALI0D05511, YALI0D01738,YALI0D02167, YALI0D04246, YALI0D05291, YALI0D07986, YALI0D10417,YALI0D14366, YALI0D25630, YALI0E03212, ALI0E07810, YALI0E12859,YALI0E14322, YALI0E15378, YALI0E15400, YALI0E18502, YALI0E18568,YALI0E22781, YALI0E25982, YALI0E28314, YALI0E32417, YALI0F01320,YALI0F06578, YALI0F07535, YALI0F14729, YALI0F22121, YALI0F25003,YALI0E14729, YALI0B17512, or a homolog of any of these. In someembodiments three, four, five, or more than five genes from this listare disrupted.

In embodiments having two disrupted genes, particularly useful genes fordisruption include, but are not limited to, the C17545 gene (or homologthereof) and/or the E30283 gene (or homolog thereof) and/or the E28336gene (or homolog thereof) and/or the E11099 gene (or homolog thereof)and/or the E28534 gene (or homolog thereof) and/or the B17512 gene (orhomolog thereof). In some embodiments, both the C17545 gene (or homologthereof) and the E28336 gene (or homolog thereof) are disrupted. In someembodiments, both the C17545 gene (or homolog thereof) and the E11099gene (or homolog thereof) are disrupted. In some embodiments, both theC17545 gene (or homolog thereof) and the E28534 gene (or homologthereof) are disrupted. In some embodiments, both the C17545 gene (orhomolog thereof) and the B17512 gene (or homolog thereof) are disrupted.In some embodiments, both the E28336 gene (or homolog thereof) and theE11099 gene are disrupted. In some embodiments, both the E28336 gene (orhomolog thereof) and the E28534 gene (or homolog thereof) are disrupted.In some embodiments, both the E28336 gene (or homolog thereof) and theB17512 gene (or homolog thereof) are disrupted. In some embodiments,both the E11099 gene (or homolog thereof) and the E28534 gene (orhomolog thereof) are disrupted. In some embodiments, both the E11099gene (or homolog thereof) and the B17512 gene (or homolog thereof) aredisrupted. In some embodiments, both the E38534 gene (or homologthereof) and the B17512 gene (or homolog thereof) are disrupted.

In one embodiment, the microbial organism, e.g. Yarrowia lipolytica, hasat least one disrupted endogenous gene that is YALI0C17545, YALI0E28336,YALI0E11099, YALI0B10406, YALI0A19536, YALI0E28534, YALI0E32769,YALI0E30283, YALI0E12463, YALI0E14729, YALI0B17512 or a homolog of anyof these. In another embodiment, the microbial organism has a firstdisrupted gene and a second disrupted gene, both selected from thisgroup of genes. In this embodiment, the microbial organism may haveadditional disrupted genes (e.g., a third, fourth, or fifth disruptedgene also selected from this group), or it may have only two disruptedgenes.

In some embodiments, the microbial organism, e.g. Yarrowia lipolytica,has two disrupted genes, or homologs thereof. In some embodiments,microbial organisms which exhibit improved production of fatty acyl-CoAderivatives comprise any of the following combinations of two disruptedendogenous genes:

a. YALI0C17545 and YALI0E28336;

b. YALI0C17545 and YALI0B10406;

c. YALI0C17545 and YALI0E28534;

d. YALI0C17545 and YALI0E30283;

e. YALI0E28336 and YALI0E30283;

f. YALI0E11099 and YALI0E30283;

g. YALI0A19536 and YALI0E30283;

h. YALI0A19536 and YALI0E28534;

i. YALI0E30283 and YALI0E12463;

j. YALI0E14729 and YALI0B10406;

k. YALI0E14729 and YALI0C17545; and

l. YALI0E14729 and YALI0E11099; and homologs of (a)-(l).

In another embodiment, the microbial organism, e.g. Yarrowia lipolytica,has three or more (e.g., 3) disrupted genes, or homologs thereof. Insome embodiments, microbial organisms which exhibit improved productionof fatty acyl-CoA derivatives comprise any of the following combinationsof three disrupted endogenous genes:

m. YALI0C17545, YALI0E28336, and YALI0E11099;

n. YALI0C17545, YALI0E28336, and YALI0B10406;

o. YALI0C17545, YALI0E28336, and YALI0A19536;

p. YALI0C17545, YALI0E28336, and YALI0E28534;

q. YALI0C17545, YALI0E28336, and YALI0E32769;

r. YALI0C17545, YALI0E28336, and YALI0E12463;

s. YALI0C17545, YALI0E11099, and YALI0B10406;

t. YALI0C17545, YALI0B10406, and YALI0A19536;

u. YALI0E28336, YALI0E11099, and YALI0B10406;

v. YALI0E11099, YALI0B10406, and YALI0A19536; and

w. YALI0C17545, YALI0E28534, and YALI0B17512; and homologs of (m)-(w).

In some embodiments, wherein the microbial organism, e.g. Yarrowialipolytica, has three or more (e.g., 3) disrupted genes, two or more ofthe disrupted genes are selected from the C17545 gene, the E28336 gene,the E11099 gene, the E28534 gene, the B17512 gene, and homologs thereof.In some embodiments, the C17545 gene (or homolog thereof) and the E28336gene (or homolog thereof) are disrupted. In some embodiments, the C17545gene (or homolog thereof) and the E11099 gene (or homolog thereof) aredisrupted. In some embodiments, the C17545 gene (or homolog thereof) andthe E28534 gene (or homolog thereof) are disrupted. In some embodiments,the C17545 gene (or homolog thereof) and the B17512 gene (or homologthereof) are disrupted. In some embodiments, the E28336 gene (or homologthereof) and the E11099 gene are disrupted. In some embodiments, theE28336 gene (or homolog thereof) and the E28534 gene (or homologthereof) are disrupted. In some embodiments, the E28336 gene (or homologthereof) and the B17512 gene (or homolog thereof) are disrupted. In someembodiments, the E11099 gene (or homolog thereof) and the E28534 gene(or homolog thereof) are disrupted. In some embodiments, the E11099 gene(or homolog thereof) and the B17512 gene (or homolog thereof) aredisrupted. In some embodiments, the E38534 gene (or homolog thereof) andthe B17512 gene (or homolog thereof) are disrupted. In some embodiments,all three of the disrupted genes are selected from the C17545 gene, theE28336 gene, the E11099 gene, the E28534 gene, the B17512 gene, andhomologs thereof. In some embodiments, the C17545 gene (or homologthereof), the E28336 gene (or homolog thereof), and the E11099 gene (orhomolog thereof) are disrupted. In some embodiments, the C17545 gene (orhomolog thereof), the E28336 gene (or homolog thereof), and the E28534gene (or homolog thereof) are disrupted. In some embodiments, the C17545gene (or homolog thereof), the E28336 gene (or homolog thereof), and theB17512 gene (or homolog thereof) are disrupted. In some embodiments, theC17545 gene (or homolog thereof), the E11099 gene (or homolog thereof),and the B17512 gene (or homolog thereof) are disrupted. In someembodiments, the C17545 gene (or homolog thereof), the E28534 gene (orhomolog thereof), and the B17512 gene (or homolog thereof) aredisrupted. In some embodiments, the E28336 gene (or homolog thereof),the E11099 gene (or homolog thereof), and the E28534 gene (or homologthereof) are disrupted. In some embodiments, the E28336 gene (or homologthereof), the E11099 gene (or homolog thereof), and the B17512 gene (orhomolog thereof) are disrupted. In some embodiments, the E28336 gene (orhomolog thereof), the E28534 gene (or homolog thereof), and the B17512gene (or homolog thereof) are disrupted. In some embodiments, the E11099gene (or homolog thereof), the E28534 gene (or homolog thereof), and theB17512 gene (or homolog thereof) are disrupted.

In yet another embodiment, the microbial organism has four or more(e.g., 4) disrupted genes, or homologs thereof. In some embodiments,microbial organisms which exhibit improved production of fatty acyl-CoAderivatives comprise any of the following combinations of four disruptedendogenous genes:

-   -   x. YALI0C17545, YALI0E28336, YALI0E11099, and YALI0B10406;    -   y. YALI0C17545, YALI0E28336, YALI0E11099, and YALI0A19536;    -   z. YALI0C17545, YALI0E28336, YALI0E11099, and YALI0E28534;    -   aa. YALI0C17545, YALI0E28336, YALI0E11099, and YALI0E32769;    -   bb. YALI0C17545, YALI0E28336, YALI0B10406, and YALI0A19536;    -   cc. YALI0C17545, YALI0E28336, YALI0B10406, and YALI0E32769;    -   dd. YALI0C17545, YALI0E28336, YALI0A19536, and YALI0E28534;    -   ee. YALI0C17545, YALI0E28336, YALI0E28534, and YALI0E32769;    -   ff. YALI0C17545, YALI0E28336, YALI0E28534, and YALI0E12463;    -   gg. YALI0E28336, YALI0E11099, YALI0B10406, and YALI0E32769;    -   hh. YALI0E11099, YALI0EA19536, YALI0B10406, and YALI0B17512; and    -   ii YALI0E11099, YALI0E28336, YALI0C17545, and YALI0E14729; and        homologs of (x)-(ii).

In some embodiments, wherein the microbial organism, e.g. Yarrowialipolytica, has four or more (e.g., 4) disrupted genes, two or more ofthe disrupted genes are selected from the C17545 gene, the E28336 gene,the E11099 gene, the E28534 gene, the B17512 gene, and homologs thereof.In some embodiments, three or more of the disrupted genes are selectedfrom the C17545 gene, the E28336 gene, the E11099 gene, the E28534 gene,the B17512 gene, and homologs thereof. In some embodiments, all four ofthe disrupted genes are selected from the C17545 gene, the E28336 gene,the E11099 gene, the E28534 gene, the B17512 gene, and homologs thereof.In some embodiments, the C17545 gene (or homolog thereof), the E28336gene (or homolog thereof), the E11099 gene (or homolog thereof), and theE28534 gene (or homolog thereof) are disrupted. In some embodiments, theC17545 gene (or homolog thereof), the E28336 gene (or homolog thereof),the E11099 gene (or homolog thereof), and the B17512 gene (or homologthereof) are disrupted. In some embodiments, the E28336 gene (or homologthereof), the E11099 gene (or homolog thereof), the E28534 gene (orhomolog thereof), and the 817512 gene (or homolog thereof) aredisrupted.

In some embodiments, any one of the endogenous genes or specificcombinations of endogenous genes listed in Table 3 or Table 4 aredisrupted in the organism. In some embodiments, the organism comprisesadditional disrupted genes. The genes recited in Table 3 and Table 4 arenamed with reference to the Yarrowia lipolytica genome; however, one ofskill in the art will recognize that equivalent disruptions can be madein a microbial organism other than Y. lipolytica (e.g., in algae,bacteria, mold, filamentous fungus, or yeast) by disrupting a homolog(s)of a gene listed in Table 3 or Table 4 in that microbial organism.

In addition to any of the endogenous gene disruptions described herein,one or more additional genes can optionally be disrupted (e.g., by“knockout,” inactivation, mutation, or inhibition as described herein),introduced, and/or modified in a microbial organism of the presentinvention. These additional genes can be, but do not need to be, genesthat have previously been identified as being involved in fatty acyl-CoAderivative production.

Methods of Disruption

As described in the definitions, the term “disrupted,” as applied to agene, refers to a genetic modification that decreases or eliminates theexpression of the gene and/or the biological activity of thecorresponding gene product (mRNA and/or protein) (e.g., for the geneslisted in Table 1, the known or predicted biological activity listed inTable 1). In some embodiments, the disruption eliminates orsubstantially reduces expression of the gene product as determined by,for example, immunoassays. “Substantially reduces,” in this context,means the amount of expressed protein is reduced by at least 50%, oftenat least 75%, sometimes at least 80%, at least 90% or at least 95%compared to expression from the undisrupted gene. In some embodiments, agene product (e.g., protein) is expressed from the disrupted gene butthe protein is mutated (e.g, a deletion of one or more amino acids, oran insertion of one or more amino acid substitutions) such that thebiological activity (e.g., enzymatic activity) of the protein iscompletely eliminated or substantially reduced. As used herein,“completely eliminated” means the gene product has no measurableactivity. “Substantially reduced,” in this context, means the biologicalactivity of the protein is reduced by at least 50%, often at least 75%,sometimes at least 80%, at least 90% or at least 95% compared to theunmutated protein. The biological activity of a gene product (e.g.,protein) can be measured by a functional assay such as an enzyme assay.For example, in some embodiments, the microbial organism has a deletionof all or a portion of the protein-encoding sequence of the endogenousgene, a mutation in the endogenous gene such that the gene encodes apolypeptide having no activity or reduced activity (e.g., insertion,deletion, point, or frameshift mutation), reduced expression due toantisense RNA or small interfering RNA that inhibits expression of theendogenous gene, or a modified or deleted regulatory sequence (e.g.,promoter) that reduces expression of the endogenous gene, any of whichmay bring about a disrupted gene. In some embodiments, all of the genesdisrupted in the microorganism are disrupted by deletion.

It will be understood that methods for gene disruption in yeast andother microorganisms are well known, and the particular method used toreduce or abolish the expression of the endogenous gene is not criticalto the invention. In one embodiment, disruption can be accomplished byhomologous recombination, whereby the gene to be disrupted isinterrupted (e.g., by the insertion of a selectable marker gene) or madeinoperative (e.g., “gene knockout”). Methods for gene knockout andmultiple gene knockout are well known. See, e.g., Example 5, infra;Rothstein, 2004, “Targeting, Disruption, Replacement, and Allele Rescue:Integrative DNA Transformation in Yeast” In: Guthrie et al., Eds. Guideto Yeast Genetics and Molecular and Cell Biology, Part A, p. 281-301;Wach et al., 1994, “New heterologous modules for classical or PCR-basedgene disruptions in Saccharomyces cerevisiae” Yeast 10:1793-1808.Methods for insertional mutagenesis are also well known. See, e.g.,Amberg et al., eds., 2005, Methods in Yeast Genetics, p. 95-100; Fickerset al., 2003, “New disruption cassettes for rapid gene disruption andmarker rescue in the yeast Yarrowia lipolytica” Journal ofMicrobiological Methods 55:727-737; Akada et al., 2006, “PCR-mediatedseamless gene deletion and marker recycling in Saccharomyces cerevisiae”Yeast 23:399-405; Fonzi et al., 1993, “Isogenic strain construction andgene mapping in Candida albicans” Genetics 134:717-728.

Antisense inhibition is well known in the art. Endogenous genes can bedisrupted by inhibiting transcription, stability, and/or translationusing antisense methods. For antisense technology, a nucleic acid strand(DNA, RNA, or analog) complementary to the gene's mRNA. is introducedinto the cell. This complementary strand will bind to the gene's mRNAand thus effectively disrupt the gene.

The method of disruption can be applied independently for each disruptedgene. Thus, when multiple genes are disrupted, the genes need not bedisrupted in the same way. For example, a microbial organism can haveone gene that is disrupted or replaced by an artificial piece of DNA(“knockout”), one gene that is disrupted by an insertion mutation, andanother gene whose promoter is altered to decrease expression. In someembodiments, two or more genes are disrupted in the same manner. In someembodiments, two or more genes are disrupted by the same disruptionevent (e.g., recombination event). In one embodiment, all of thedisrupted genes are disrupted in the same manner or by the samedisruption event. In one embodiment, all of the disrupted genes are“knockout” genes, that is, genes that are inactivated by disrupting orreplacing at least a portion of the coding sequence. In anotherembodiment, all of the disrupted genes are knockout genes that aredisrupted by the same disruption event.

In one embodiment, multiple gene copies are disrupted. A “gene copy,” asused herein, refers to the same target gene (e.g., an endogenous gene asdescribed herein) on a homologous chromosome in a diploid or polyploidorganism. For example, a microbial organism may have multiple sets ofchromosomes and thus possess multiple copies of each target gene. Insome embodiments, a microbial organism is diploid (i.e., having two setsof chromosomes and thus two copies of each target gene). In someembodiments, a microbial organism is polyploid (i.e., having more thantwo sets of chromosomes). In some embodiments, a microbial organism istriploid (i.e., having three sets of chromosomes and thus three copiesof each target gene). In some embodiments, a microbial organism istetraploid (i.e., having four sets of chromosomes and thus four copiesof each target gene). In some embodiments, a microbial organism has 2,3, 4, 5, 6, 7, 8, 9, 10, or more copies of a target gene. In someembodiments, the microbial organism possesses 2, 3, 4, 5, 6, 7, 8, 9,10, or more disrupted copies of a target endogenous gene. In oneembodiment, all copies of the target endogenous gene are disrupted inthe microbial organism.

The term “one or more gene copies” refers to the number of copies of thesame target gene, while “one or more disrupted genes” refers to one ormore individual genes. For example, a microbial organism can have twodisrupted gene copies while having only one disrupted gene.

Where two or more endogenous genes are disrupted, the number of copiesto be disrupted can be selected independently for each disrupted gene.Multiple copies of a gene can be disrupted by, e.g., performing multiplerounds of recombination with a recoverable marker.

IV. Expression of Truncated Sec62

In another aspect, the invention relates to recombinant microbialorganisms, such as yeast, in which an endogenous gene encoding a Sec62protein, or a homolog or allelic variant thereof, has been modified.Sec62 is a protein that is involved in the translocation of proteinsinto the endoplasmic reticulum in yeast. Yarrowia Sec62 is encoded byYALI0B17512, has the amino acid sequence set forth as SEQ ID NO:64, andcontains a cytoplasmic domain (amino acids 207 to 396 of SEQ ID NO:64).See also GenBank Accession No. CAA67878.1 and Swennen et al., 1997,“Cloning the Yarrowia lipolytica homologue of the Saccharomycescerevisiae SEC62 gene,” Curr Genet. 31(2):128-132. As described in theexample below, we have discovered that yeast cells expressing atruncated Sec62 protein which lacks a complete cytoplasmic domain haveincreased production of fatty acyl-CoA derivatives as compared to acontrol yeast cell in which the Sec62 protein is not truncated.

Thus, the invention provides a microbial organism expressing a truncatedSec62 protein or homolog. The organism can be used for any of themethods or processes described herein, and may be combined withdisrupted genes described herein and in combinations described herein.

Thus, in some embodiments, the organism, e.g. an algae, a bacteria, amold, a filamentous fungus, or a yeast (e.g., Yarrowia lipolytica), isone in which the endogenous gene encoding Sec62 (YALI0B17512 or ahomolog thereof) comprises a partial deletion of the sequence encodingat least a portion of the cytoplasmic domain of the encoded Sec62protein. In some embodiments, the partial deletion of the codingsequence comprises a deletion of the entire cytoplasmic domain of theencoded Sec62 protein.

In some embodiments, the Sec62 protein is SEQ ID NO:64 or is a homologor allelic variant substantially identical to SEQ ID NO:64 (e.g, has asequence identity of at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ IDNO:64). In some embodiments, the Sec62 protein is isolated or derivedfrom an organism selected from the group consisting of Saccharomycescerevisiae (Genbank Accession No. CAB56541.1; SEQ ID NO:77),Kluyveromyces lactis (Genbank Accession No. CAH00127.1; SEQ ID NO:78),and Schizosaccharomyces pombe (Genbank Accession No. CAB16220.1; SEQ IDNO:79).

In some embodiments, the microbial organism (e.g., Y. lipolytica)expresses a truncated Sec62 protein or homolog in which the entirecytoplasmic domain (corresponding to amino acids 207-396 of SEQ IDNO:64) has been deleted. In some embodiments, the microbial organismexpresses a truncated Sec62 protein or homolog in which a portion of thecytoplasmic domain is deleted, e.g., from about position 210 to aboutposition 396; from about position 250 to about position 396; from aboutposition 300 to about position 396; from about position 330 to aboutposition 396; from about position 210 to about position 350; from aboutposition 210 to about position 300; from about position 250 to aboutposition 350; or from about position 300 to about position 350, whereinthe amino acids are numbered with reference to SEQ ID NO:64. In someembodiments, the microbial organism expresses a truncated Sec62 proteinor homolog in which a portion of the cytoplasmic domain from aboutposition 267 to about position 396 is deleted. In some embodiments, themicrobial organism expresses a truncated Sec62 protein or homolog inwhich a portion of the cytoplasmic domain from about position 302 toabout position 396 is deleted. In some embodiments, the microbialorganism expresses a truncated Sec62 protein or homolog in which aportion of the cytoplasmic domain from about position 337 to aboutposition 396 is deleted.

In some embodiments, a microbial organism is diploid (i.e., having twosets of chromosomes and thus two copies of the gene encoding Sec62). Insome embodiments, a microbial organism is polyploid (i.e., having morethan two sets of chromosomes and thus more than two copies of the geneencoding Sec62). In some embodiments, more than one copy of the Sec62gene is modified to express a truncated Sec62 protein. In someembodiments, all of the copies of the Sec62 gene are modified to expressa truncated Sec62 protein.

It will be understood that the particular method used to delete all or aportion of the cytoplasmic domain of Sec62 is not critical to theinvention. In some embodiments, deletion of the cytoplasmic domain orportion thereof can be accomplished by replacing the portion of thesequence that encodes the cytoplasmic domain or portion thereof with anartificial piece of DNA (e.g., a selectable marker). In someembodiments, deletion of the cytoplasmic domain or portion thereof canbe accomplished by removing the portion of the coding sequence thatencodes the cytoplasmic domain or portion thereof.

V. Exogenous Far Expression

FAR Protein

In one aspect, the modified microbial organism exhibiting improvedproduction of fatty acyl-CoA derivatives (e.g., a microbial organism,such as Yarrowia lipolytica, in which one, two, three, four, or moreendogenous genes is disrupted as described herein) expresses oroverexpresses a FAR. As described in the Examples section, microbialorganisms in which certain endogenous genes or combinations of genes aredisrupted and which express an exogenous gene encoding a FAR proteinhave increased production of fatty acyl-CoA derivatives, as compared tocontrol microbial organisms (e.g., otherwise identical microbialorganisms) expressing the exogenous gene encoding the FAR protein inwhich the corresponding endogenous genes have not been disrupted.

In some embodiments, the organism, e.g. an algae, a bacteria, a mold, afilamentous fungus, or a yeast (e.g., Yarrowia lipolytica), expresses anexogenous FAR protein (i.e., a FAR not normally expressed in theorganism, such as a protein derived from a different species). In someembodiments, the exogenous FAR protein is a wild-type FAR protein. Insome embodiments, the exogenous FAR protein is selected or engineeredfor increased activity or yield of fatty acyl-CoA derivatives, e.g.,fatty alcohols (i.e., a FAR variant as described herein). In someembodiments, the FAR protein is a FAR protein or variant as described inUS patent publication 2011/0000125 or in U.S. patent application Ser.No. 13/171,138, filed Jun. 28, 2011, the entire contents of each ofwhich are incorporated herein by reference.

In one embodiment, the exogenous FAR protein is from a genus of marinebacteria such as gammaproteobacteria (e.g., Marinobacter andOceanobacter). In one embodiment, the exogenous FAR protein is from aspecies of the genus Marinobacter including, but not limited to, M.aquaeolei, M. arcticus, M. actinobacterium, and M. lipolyticus. In oneembodiment, the exogenous FAR protein is from M. algicola (also referredto herein as “FAR_Maa”). In one embodiment, the exogenous FAR protein isfrom M. aquaeolei (also referred to herein as “FAR_Maq”). In anotherembodiment, the exogenous FAR protein is from a species of the genusOceanobacter including, but not limited to, Oceanobacter sp. Red65(renamed Bermanella marisrubi) (also referred to herein as “FAR_Ocs”),Oceanobacter strain WH099, and O. kriegii. In another embodiment, theexogenous FAR protein is from Hahella including, but not limited to, H.chejuensis and equivalent species thereof.

In one embodiment, the exogenous FAR gene is FAR_Maa (wild-type FAR fromMarinobacter algicola strain DG893, SEQ ID NO:1), FAR_Maq (wild-type FARfrom Marinobacter aquaeolei, SEQ ID NO:3), FAR_Ocs (wild-type FAR fromOceanobacter sp. RED65, SEQ ID NO:5), or a fragment that encodes afunctional FAR enzyme. In one embodiment, the FAR gene has a DNAsequence identity of at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% to any of SEQ ID NOs:1,3, or 5. In one embodiment, the FAR gene has a DNA sequence identity ofat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% to SEQ ID NO:1.

In another embodiment, the exogenous FAR protein has a sequence identityof at least 80%, at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% to any of SEQ ID NOs:2, 4, or 6, whichcorrespond to the polypeptide sequences of wild-type FAR_Maa, wild-typeFAR_Maq, and wild-type FAR_Ocs, respectively. In one embodiment, the FARprotein has a sequence identity of at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ IDNO:2.

In other embodiments, the FAR enzyme is FAR_Hch (Hahella chejuensis KCTC2396, GenBank No. YP_(—)436183.1, SEQ ID NO:65), FAR_Mac (from marineactinobacterium strain PHSC20C1, SEQ ID NO:66), FAR_JVC(JCVI_ORF_(—)1096697648832, GenBank No. EDD40059.1, SEQ ID NO:67),FAR_Fer (JCVI_SCAF_(—)1101670217388, SEQ ID NO:68), FAR_Key(JCVLSCAF_(—)1097205236585, SEQ ID NO:69), FAR_Gal(JCVLSCAF_(—)1101670289386, SEQ ID NO:70), or a variant or functionalfragment thereof. Table 2 provides the approximate amino acid sequenceidentity of these bacterial FAR proteins to FAR_Maa (SEQ ID NO:2) andFAR_Ocs (SEQ ID NO:6).

TABLE 2 Amino acid sequence identity of homologs relative to FAR_Maa andFAR_Ocs % Sequence Identity % Sequence Identity to FAR_Maa to FAR_OcsFAR Gene (SEQ ID NO: 2) (SEQ ID NO: 6) FAR_Maa 100 46 FAR_Mac 32 31FAR_Fer 61 36 FAR_Gal 25 25 FAR_JVC 34 30 FAR_Key 32 30 FAR_Maq 78 45FAR_Hch 54 47

In other embodiments, the FAR enzyme or functional fragment is isolatedor derived from an organism selected from the group consisting of Vitisvinifera (GenBank Accession No. CA022305.1, SEQ ID NO:71; or CA067776.1,SEQ ID NO:72), Desulfatibacillum alkenivorans (GenBank Accession No.NZ_ABI101000018.1), Stigmatella aurantiaca (NZ_AAMD01000005.1, SEQ IDNO:73), and Phytophthora ramorum (GenBank Accession No.:AAQX01001105.1).

FAR Variants

In some embodiments, variants of FAR enzymes are used, such asfunctional fragments and variants selected using molecular evolutiontechnology. A “functional fragment,” as used herein, refers to apolypeptide having an amino-terminal and/or carboxy-terminal deletionand/or internal deletion, but in which the remaining amino acid sequenceis identical or substantially identical to the corresponding positionsin the sequence to which it is being compared (e.g., a full-lengthwild-type FAR protein or full-length FAR variant protein) and whichretains substantially all (e.g., retains at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or more) of the activity of thefull-length polypeptide (e.g., the full-length wild-type FAR protein orfull-length FAR variant protein). Functional fragments can comprise upto 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of thefull-length FAR protein. Thus, a functional fragment, in this context,is a fragment of a naturally occurring FAR polypeptide, or variantthereof, that has catalytic activity. In some embodiments, thefunctional fragment has at least 50% of the activity of thecorresponding full-length wild-type FAR from which it is derived (e.g.,FAR_Maa, FAR_Maq, or FAR_Ocs).

In some embodiments, a FAR variant comprises one or more mutations(e.g., substitutions) as compared to a wild-type FAR, such that theresulting FAR variant polypeptide has improved characteristics and/orproperties as compared to the wild-type FAR, such as, for example,increased fatty alcohol production when the FAR variant is expressed ina host cell. In some embodiments, a variant FAR protein may have from 1to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, or more amino acid substitutionsrelative to a native (wild-type) FAR protein such as FAR_Maa (SEQ IDNO:2), FAR_Maq (SEQ ID NO:4), or FAR_Ocs (SEQ ID NO:6). In someembodiments, a variant FAR protein may have from 1 to 50, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, or more amino acid substitutions relative to the native FARprotein of SEQ ID NO:2. In some embodiments, a variant FAR protein mayhave from 1 to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or more amino acidsubstitutions relative to the native FAR protein of SEQ ID NO:4. In someembodiments, a variant FAR protein may have from 1 to 50, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, or more amino acid substitutions relative to the native FARprotein of SEQ ID NO:6.

In some embodiments, a FAR variant comprises at least about 70% (or atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%) sequence identity to awild-type FAR (e.g., a FAR polypeptide of SEQ ID NO:2, SEQ ID NO:4, orSEQ ID NO:6) and further comprises one or more amino acid substitutions(e.g., e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, or more amino acid substitutions)relative to the wild-type FAR, and is capable of producing at leastabout 1.5-fold, at least about 2-fold, at least about 3-fold, at leastabout 4-fold, at least about 5-fold, at least about 6-fold, at leastabout 7-fold, at least about 8-fold, at least about 9-fold, or at leastabout 10-fold more fatty alcohol than the wild-type FAR from which it isderived when assayed under the same conditions.

In certain embodiments, the microbial organism does not express anendogenous FAR (i.e., the genome of the wild-type organism does notencode a FAR). In some embodiments, the microbial organism is anorganism that expresses an endogenous FAR protein. In certainembodiments, the microbial organism is an organism that does not expressan exogenous FAR protein. In some embodiments, the microbial organism isan organism that expresses neither an endogenous FAR protein nor anexogenous FAR protein. In some embodiments, the microbial organismexpresses both endogenous FAR(s) and exogenous FAR(s).

Methods for introducing exogenous genes (e.g., FAR encoding genes) intoa host organism and expressing an exogenous protein are known in theart. See Section VII below.

VI. Microbial Organisms

Host Cells

The microbial organism in which one or more endogenous genes aredisrupted, and which exhibits improved production of fatty acyl-CoAderivatives, can be any “host cell” that produces fatty acyl-CoAderivatives. Suitable host cells include, but are not limited to, algae,bacteria, mold, filamentous fungus, and yeast, including oleaginousyeast (e.g., Yarrowia lipolytica). In some embodiments, the microbialorganism is an oleaginous organism, e.g., an organism that tends tostore its energy source in the form of oil. The host cell can beeukaryotic or prokaryotic.

In one embodiment, the microbial organism is a fungus. Suitable fungalhost cells include, but are not limited to, Ascomycota, Basidiomycota,Deuteromycota, Zygomycota, Fungi imperfecti. Particularly preferredfungal host cells are yeast cells and filamentous fungal cells.

In one embodiment, the microbial organism is a yeast. In one embodiment,the yeast is from one of the genera: Yarrowia, Brettanomyces, Candida,Cryptococcus, Endomycopsis, Hansenula, Kluyveromyces, Lipomyces,Pachysolen, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces,Schizosaccharomyces, Trichosporon, or Trigonopsis. In one embodiment,the yeast is from the genus Yarrowia. In some embodiments of theinvention, the yeast cell is Hansenula polymorpha, Saccharomycescerevisiae, Saccaromyces carlsbergensis, Saccharomyces diastaticus,Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomycespombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichiakodamae, Pichia membranaefaciens, Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi,Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyceslactis, Candida albicans, and Yarrowia lipolytica.

In one embodiment, the microbial organism is an oleaginous yeast.Oleaginous yeasts accumulate lipids such as tri-acyl glycerols. Examplesof oleaginous yeast include, but are not limited to, Yarrowialipolytica, Yarrowia paralipolytica, Candida revkauji, Candidapulcherrima, Candida tropicalis, Candida utilis, Candida curvata D,Candida curvataR, Candida diddensiae, Candida boldinii, Rhodotorulaglutinous, Rhodotorula graminis, Rhodotorula mucilaginosa, Rhodotorulaminuta, Rhodotorula bacarum, Rhodosporidium toruloides, Cryptococcus(terricolus) albidus var. albidus, Cryptococcus laurentii, Trichosporonpullans, Trichosporon cutaneum, Trichosporon cutancum, Trichosporonpullulans, Lipomyces starkeyii, Lipomyces lipoferus, Lipomycestetrasporus, Endomycopsis vernalis, Hansenula ciferri, Hansenulasaturnus, and Trigonopsis variabilis.

In one embodiment, the yeast is Yarrowia lipolytica. Exemplary Yarrowialipolytica strains include, but are not limited to, DSMZ 1345, DSMZ3286, DSMZ 8218, DSMZ 70561, DSMZ 70562, DSMZ 21175 available from theDeutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, and alsostrains available from the Agricultural Research Service (NRRL) such asbut not limited to NRRL YB-421, NRRL YB-423, NRRL YB-423-12 and NRRLYB-423-3.

In one embodiment, the host cell is a filamentous fungus. Thefilamentous fungal host cells of the present invention include allfilamentous forms of the subdivision Eumycotina and Oomycota (Hawksworthet al., 1995, in Ainsworth and Bisby's Dictionary of The Fungi, 8thed.). Filamentous fungi are characterized by a vegetative mycelium witha cell wall composed of chitin, cellulose, and other complexpolysaccharides. As used herein, the filamentous fungal host cells ofthe present invention are morphologically distinct from yeast. Exemplaryfilamentous fungal cells include, but are not limited to, species ofAchlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera,Ceriporiopsis, Cephalosporium, Chtysosporium, Cochliobolus, Corynascus,Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis,Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora,Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces,Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium,Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes,Tolypocladium, Trichoderma, Verticillium, Volvariella, includingteleomorphs, anamorphs, synonyms, basionyms, and taxonomic equivalentsthereof.

In some embodiments, the host cell is an algal cell such asChlamydomonas (e.g., C. Reinhardtii) and Phormidium (P. sp. ATCC29409).

Suitable prokaryotic cells include gram positive, gram negative andgram-variable bacterial cells. Exemplary prokaryotic host cells include,but are not limited to, species of Agrobacterium, Alicyclobacillus,Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter,Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio,Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium,Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter,Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium,Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus,Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium,Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea,Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas,Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus,Streptomyces, Streptococcus, Synecoccus, Saccharomonospora,Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium,Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus,Ureaplasma, Xanthomonas, Xylella, Yersinia, and Zymomonas. In someembodiments, the host cell is a species of Agrobacterium, Acinetobacter,Azobacter, Bacillus, Bifidobacterium, Buchnera, Geobacillus,Campylobacter, Clostridium, Corynebacterium, Escherichia, Enterococcus,Erwinia, Flavobacterium, Lactobacillus, Lactococcus, Pantoea,Pseudomonas, Staphylococcus, Salmonella, Streptococcus, Streptomyces, orZymomonas.

Transformation and Cell Culture

In another embodiment, the invention provides a method comprisingproviding a microbial organism as described herein, and culturing themicrobial organism under conditions in which fatty acyl-CoA derivativesare produced. In some embodiments, the microbial organism having one ormore disrupted endogenous genes is capable of improved production asdescribed above, e.g., at least a 1-fold increase in the production offatty acyl-CoA derivatives compared to a control organism of the sametype (e.g., an otherwise identical control microbial organism in whichthe one or more genes are not disrupted).

In some embodiments, a polynucleotide encoding a FAR polypeptide (e.g.,a wild-type FAR polypeptide or a FAR variant polypeptide) is introducedinto the microbial organism for expression of the wild-type FARpolypeptide or FAR variant polypeptide. The polynucleotide may beintroduced into the cell as a self-replicating episome (e.g., expressionvector) or may be stably integrated into the host cell DNA.

Methods, reagents, and tools for transforming microbial organismsdescribed herein, such as bacteria, yeast (including oleaginous yeast)and filamentous fungi are known in the art. General methods, reagentsand tools for transforming, e.g., bacteria can be found, for example, inSambrook et al (2001) Molecular Cloning: A Laboratory Manual, 3^(rd)ed., Cold Spring Harbor Laboratory Press, New York. Methods, reagentsand tools for transforming yeast are described in “Guide to YeastGenetics and Molecular Biology,” C. Guthrie and G. Fink, Eds., Methodsin Enzymology 350 (Academic Press, San Diego, 2002). Methods, reagentsand tools for transforming, culturing, and manipulating Y. lipolyticaare found in “Yarrowia lipolytica,” C. Madzak, J. M. Nicaud and C.Gaillardin in “Production of Recombinant Proteins. Novel Microbial andEucaryotic Expression Systems,” G. Gellissen, Ed. 2005, which isincorporated herein by reference for all purposes. In some embodiments,introduction of the DNA construct or vector of the present inventioninto a host cell can be effected by calcium phosphate transfection,DEAE-Dextran mediated transfection, PEG-mediated transformation,electroporation, or other common techniques (See Davis et al., 1986,Basic Methods in Molecular Biology, which is incorporated herein byreference).

The microbial organisms can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants, or amplifying the FAR polynucleotide. Culture conditions,such as temperature, pH and the like, will be apparent to those skilledin the art. As noted, many references are available for the culture andproduction of many cells, including cells of bacterial, plant, animal(especially mammalian) and archebacterial origin. See e.g., Sambrook,Ausubel, and Berger (all supra), as well as Freshney (1994) Culture ofAnimal Cells, a Manual of Basic Technique, third edition, Wiley-Liss,New York and the references cited therein; Doyle and Griffiths (1997)Mammalian Cell Culture: Essential Techniques John Wiley and Sons, NY;Humason (1979) Animal Tissue Techniques, fourth edition W.H. Freeman andCompany; and Ricciardelli, et al., (1989) In Vitro Cell Dev. Biol.25:1016-1024, all of which are incorporated herein by reference. Forplant cell culture and regeneration, Payne et al. (1992) Plant Cell andTissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.;Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;Fundamental Methods Springer Lab Manual, Springer-Verlag (BerlinHeidelberg New York); Jones, ed. (1984) Plant Gene Transfer andExpression Protocols, Humana Press, Totowa, N.J. and Plant MolecularBiology (1993) R. R. D. Croy, Ed. Bios Scientific Publishers, Oxford,U.K. ISBN 0 12 198370 6, all of which are incorporated herein byreference. Cell culture media in general are set forth in Atlas andParks (eds.) The Handbook of Microbiological Media (1993) CRC Press,Boca Raton, Fla., which is incorporated herein by reference. Additionalinformation for cell culture is found in available commercial literaturesuch as the Life Science Research Cell Culture Catalogue (1998) fromSigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-LSRCCC”) and, for example,The Plant Culture Catalogue and supplement (1997) also fromSigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-PCCS”), all of which areincorporated herein by reference.

VII. Additional Metabolic Engineering

In one embodiment, the modified microbial organism exhibiting improvedproduction of fatty acyl-CoA derivatives contains an exogenous geneoperably linked to a promoter that is functional in the microbialorganism. The incorporation of an exogenous gene (e.g., a FAR gene asdescribed above) can be accomplished by techniques well known in theart.

In some embodiments, the microbial organism can be modified to expressor over-express one or more genes encoding enzymes, other than FAR, thatare involved in fatty acyl-CoA derivative biosynthesis. See FIG. 1. Inparticular embodiments, the gene encodes a fatty acid synthase (FAS), anester synthase, an acyl-ACP thioesterase (TE), a fatty acyl-CoA synthase(FACS), or an acetyl-CoA carboxylase (ACC). For example, in oneembodiment, the microbial organism can be modified to express an estersynthase to produce fatty esters. Similarly, in another embodiments, themicrobial organism can be modified to express thioestersase to producefatty acids. Any of these exemplary genes can be used instead of, or inaddition to, FAR. When multiple exogenous genes are expressed, in someembodiments, the expression vector encoding a first enzyme (e.g., FAR)and the expression vector encoding a second enzyme (e.g., an FAS, estersynthase, TE, FACS, or ACC) are separate nucleic acids. In otherembodiments, the first enzyme and the second enzyme are encoded on thesame expression vector, and expression of each enzyme is independentlyregulated by a different promoter.

As shown in FIG. 1, the various fatty acyl-CoA derivatives may beproduced by the microbial organism. When recovery of a particularderivative is desired, the expression or activity of one or more of thepolypeptides involved in this metabolic pathway can be altered topreferentially yield the desired derivative. For example, one can modifythe expression or activity of one, or more of acetyl-CoA carboxylase,pyruvate decarboxylase, isocitrate dehydrogenase, ATP-citrate lyase,malic enzyme, AMP-deaminase, glucose-6-phosphate dehydrogenase,6-phosphogluconate dehydrogenase, fructose 1,6 bisphosphatase, NADHkinase, transhydrogenase, acyl-CoA:diacylglycerol acyltransferase,phospholipid:diacylglycerol acyltransferase, acyl-CoA: cholesterolacyltransferase, triglyceride lipase, and acyl-coenzyme A oxidase.

As another example, the microbial organism can be modified to utilizeparticular desired substrates. For example, although wild-type Y.lipolytica does not preferentially utilize xylose as a substrate, it canbe genetically engineered to do so. See, e.g., Brat et al., 2009,“Functional expression of a bacterial xylose isomerase in Saccharomycescerevisiae” Applied and Environmental Microbiology 75:2304-11; Ho etal., 1998, “Genetically engineered Saccharomyces yeast capable ofeffective cofermentation of glucose and xylose” Applied andEnvironmental Microbiology 64:1852-59. Similarly, Y. lipolytica may alsobe engineered to utilize sucrose. See, e.g., Nicaud et al., 1989,“Expression of invertase activity in Yarrowia lipolytica and its use asa selectable marker” Current Genetics 16:253-260. It may be advantageousto engineer the microbial organisms to be tailored to, particularenvironmental conditions, for example, to utilize feedstock obtainedfrom a cellulosic or lignocellulosic biomass wherein the feedstock maybe contacted with cellulase enzymes to provide fermentable sugarsincluding but not limited to glucose, fructose, xylose, and sucrose.

In some embodiments, a microbial organism as described herein (e.g., amicrobial organism comprising one or more disrupted endogenous genesselected from YALI0C17545, YALI0E28336, YALI0E11099, YALI0B10406,YALI0A19536, YALI0E28534, YALI0E32769, YALI0E30283, YALI0E12463,YALI0E17787, YALI0B14014, YALI0A10769, YALI0A15147, YALI0A16379,YALI0A20944, YALI0B07755, YALI0B10175, YALI0B13838, YALI0C02387,YALI0D05511, YALI0D01738, YALI0D02167, YALI0D04246, YALI0D05291,YALI0D07986, YALI0D10417, YALI0D14366, YALI0D25630, YALI0E03212,ALI0E07810, YALI0E12859, YALI0E14322, YALI0E15378, YALI0E15400,YALI0E18502, YALI0E18568, YALI0E22781, YALI0E25982, YALI0E28314,YALI0E32417, YALI0F01320, YALI0F06578, YALI0F07535, YALI0F14729,YALI0F22121, YALI0F25003, YALI0E14729, YALI0B17512, and homologsthereof, and an exogenous gene encoding an exogenous FAR operably linkedto a promoter) further comprises an exogenous gene encoding an enzymethat catalyzes the hydrolysis of a fermentable sugar (e.g., sucrose,arabinose, or mannose). Examples of enzymes that catalyze the hydrolysisof a fermentable sugar include, but are not limited to, sucrases andinvertases. Thus, in some embodiments, the exogenous gene encodes asucrase or an invertase. In some embodiments, the exogenous gene is aSUC2 gene, which encodes invertase. Invertases (EC 3.2.1.26) catalyzethe hydrolysis of sucrose resulting in a mixture of glucose andfructose. Sucrases are related to invertases but catalyze the hydrolysisof sucrose by a different mechanism.

The exogenous gene encoding the enzyme that catalyzes the hydrolysis ofa fermentable sugar may be derived from any suitable microbial organism,e.g., from algae, bacteria, mold, filamentous fungus, or yeast. In someembodiments, the microbial organism comprising one or more disruptedendogenous genes is Y. lipolytica, and the exogenous gene encoding anenzyme that catalyzes the hydrolysis of sucrose is from Saccharomycescerevisiae. In some embodiments, the exogenous gene is Saccharomycescerevisiae SUC2 invertase.

Targeted Integration of an Exogenous Gene

In some embodiments, expression of an exogenous gene in the microbialorganism is accomplished by introducing the exogenous gene into theorganism on an episomal plasmid. In some embodiments, expression of theexogenous gene is accomplished by integrating the gene into the genomeof the microbial organism. Integration of the exogenous gene into thegenome of the microbial organism has various advantages over the use ofplasmids, including but not limited to less variation in proteinexpression, greater flexibility in the choice of fermentation media, andthe potential for high levels of expression by introducing multiplecopies of a single gene.

Thus, in some embodiments, a microbial organism having one or moredisrupted endogenous genes as described herein further comprises anexogenous gene encoding an enzyme that is involved in fatty acyl-CoAderivative biosynthesis (e.g., a FAR enzyme), wherein the exogenous geneis integrated into the genome of the microbial organism. In someembodiments, the microbial organism comprises an exogenous gene encodinga FAR protein (e.g., a wild-type FAR protein that is identical orsubstantially identical to the FAR polypeptide of any of SEQ ID NOs:2,4,or 6, or a FAR variant protein as described herein) that is integratedinto the genome of the microbial organism.

In some embodiments, the microbial organism comprises one copy of theexogenous gene. In some embodiments, the microbial organism comprisestwo, three, four, five, or more copies of the exogenous gene. In someembodiments, multiples copies of the exogenous gene (e.g., two, three,four, five, or more copies) are integrated into the genome of themicrobial organism in a direct repeat structure or an inverted repeatstructure.

In some embodiments, integration of the exogenous gene into the genomeof the microbial organism may be targeted to one or more particularregions of the microbial genome. The genome of the microbial organismcan be mapped to identify regions wherein integration of an exogenousgene results in improved expression of the gene, or an improved property(e.g., improved fatty alcohol production) relative to the expression ofthe exogenous gene in a control organism of the same type (e.g., anotherwise identical organism) by a plasmid (also called “hotspots” ofexpression). As shown below in the Examples, following integration of anexogenous gene encoding the FAR protein into a Y. lipolytica strain,strains were identified that showed particularly good improvement infatty alcohol production relative to a Y. lipolytica strain thatexpressed FAR via plasmid. These integration hotspots of expression,once mapped, can then be targeted for subsequent integration of anexogenous gene via homologous recombination.

Thus, in some embodiments, the exogenous gene is integrated into achromosomal site in the genome of the microbial organism that is ahotspot of expression. In some embodiments, wherein the microbialorganism is Y. lipolytica, the exogenous gene is integrated into thegenome of the microbial organism at one or more of the chromosomal sitesdescribed herein, for example in Example 1.

Targeted integration of an exogenous gene into the genome of a microbialorganism of the present invention can also be accomplished via“seamless” marker recycling. As described in the Examples section below,in seamless marker recycling a bifunctional selectable marker isintroduced into a specific genomic location, either to disrupt a nativegene or to introduce an exogenous gene. Integrants are identified usingthe selectable marker (positive selection, e.g., using a marker thatconfers antibiotic resistance). The marker is then excised, or“recycled,” via homologous recombination between two flanking repeats,and organisms that have successfully recycled the marker are identifiedby counter-selection (negative selection, e.g., using a marker thatinduces toxicity). The selectable marker can then be used again tointroduce additional modifications into the genome of the organism. Thismethod is advantageous because it permits a theoretically unlimitednumber of targeted modifications (e.g., targeted deletions of genes ortargeted integrations of exogenous genes) to be made to the genome of anorganism, thus facilitating strain development.

Thus, in some embodiments, an exogenous gene (e.g., a gene encoding anenzyme that is involved in fatty acyl-CoA derivative biosynthesis, e.g.,a FAR enzyme) is integrated into the genome of a microbial organism ofthe present invention (e.g., a microbial organism having one or moredisrupted endogenous genes as described herein) using a recyclablebifunctional selectable marker having a positive selectable marker and anegative selectable marker, wherein integration of the exogenous geneinto the genome is identified using the positive selectable marker andwherein subsequent recycling of the bifunctional marker is identifiedusing the negative selectable marker. In some embodiments, thebifunctional selectable marker has a hygromycin positive selectablemarker and a thymidine kinase negative selectable marker.

Vectors

Expression vectors may be used to transform a microbial organism of thepresent invention (e.g., a microbial organism having one or moredisrupted endogenous genes as described herein) with a gene encoding aFAR enzyme, and/or a gene encoding an enzyme other than FAR that isinvolved in fatty acyl-CoA derivative biosynthesis, and/or a geneencoding an enzyme that catalyzes the hydrolysis of a fermentable sugar.A recombinant expression vector can be any vector, e.g., a plasmid or avirus, which can be manipulated by recombinant DNA techniques tofacilitate expression of the exogenous gene in the microbial organism.In some embodiments, the expression vector is stably integrated into thechromosome of the microbial organism. In other embodiments, theexpression vector is an extrachromosomal replicative DNA molecule, e.g.,a linear or closed circular plasmid, that is found either in low copynumber (e.g., from about 1 to about 10 copies per genome equivalent) orin high copy number (e.g., more than about 10 copies per genomeequivalent).

Expression vectors for expressing the one or more exogenous genes arecommercially available, e.g., from Sigma-Aldrich Chemicals, St. Louis,Mo. and Stratagene, LaJolla, Calif. In some embodiments, examples ofsuitable expression vectors are plasmids which are derived from pBR322(Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly (Latheet al., 1987, Gene 57:193-201).

In some embodiments, an expression vector optionally contains a ribosomebinding site (RBS) for translation initiation, and a transcriptionterminator, such as PinII. The vector also optionally includesappropriate sequences for amplifying expression, e.g., an enhancer.

In particular embodiments, the present disclosure provides an autonomousreplicating plasmid for expression of exogenous genes in Yarrowia, andparticularly in Y. lipolytica. An exemplary plasmid is shown in FIG. 2and described in the Examples. Such a plasmid can be further modifiedfor expression of exogenous genes useful for fatty acyl-CoA derivativeproduction in yeast, inter alia, Y. lipolytica.

In some embodiments, wherein more than one exogenous gene is to beexpressed in the microbial organism (e.g., a first exogenous geneencoding a wild-type FAR polypeptide or a FAR variant polypeptide, and asecond exogenous gene encoding an enzyme other than FAR that is involvedin fatty acyl-CoA derivative biosynthesis or an enzyme that catalyzesthe hydrolysis of a fermentable sugar), the expression vector encodingthe FAR polypeptide and the expression vector encoding the second enzymeare separate nucleic acids. In other embodiments, the FAR polypeptideand the second enzyme are encoded on the same expression vector, andexpression of each enzyme is independently regulated by a differentpromoter.

Promoters

The promoter sequence is a nucleic acid sequence that is recognized by ahost cell for expression of a polynucleotide, such as a polynucleotidecontaining the coding region. Generally, the promoter sequence containstranscriptional control sequences, which mediate expression of thepolynucleotide. The promoter may be any nucleic acid sequence that showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell. Methods for the isolation, identificationand manipulation of promoters of varying strengths are available in orreadily adapted from the art. See, e.g., Nevoigt et al. (2006) Appl.Environ. Microbiol. 72:5266-5273, the disclosure of which is hereinincorporated by reference in its entirety.

In a yeast host, useful promoters include, but are not limited to thosefrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae galactokinase (GALI), Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.Exemplary Y. lipolytica promoters include, but are not limited to, TEF1,RPS7 (Müller et al., 1998, “Comparison of expression systems in theyeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyceslactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning oftwo novel promoters from Yarrowia lipolytica” Yeast 14:1267-1283), GPD,GPM (U.S. Pat. No. 7,259,255), GPAT (U.S. Pat. No. 7,264,949), FBA1(U.S. Pat. No. 7,202,356), the Leu2 promoter and variants thereof (U.S.Pat. No. 5,786,212), the EF1alpha protein promoter (WO 97/44470), Xpr2(U.S. Pat. No. 4,937,189), Tefl, Caml (YALI0C24420g), YALI0DI6467g, Tef4(YALI0BI2562g), Yef3 (YALI0E13277g), Pox2, Yat1 (US 2005/0130280),promoters disclosed in US 2004/0146975 and U.S. Pat. No. 5,952,195,CYP52A2A (US 2002/0034788); sequences from fungal (e.g., C. tropicalis)catalase, citrate synthase, 3-ketoacyl-CoA thiolase A, citrate synthase,O-acetylhornserine sulphydrylase, protease, camitineO-acetyltransferase, hydratasedehydrogenase, epimerase genes; Pox4 genes(US 2004/0265980); and Met2, Met3, Met6, Met25, and YALI 0D12903g genes.See also WO 2008/042338. Other useful promoters for yeast host cells aredescribed by Romanos et al., 1992, “Foreign gene expression in yeast: areview” Yeast 8:423-488.

For bacterial host cells, suitable promoters include, but are notlimited to, promoters obtained from the E. coli lac operon, Streptomycescoelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene(sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xylA and xylB genes,Bacillus megaterium promoters, and prokaryotic beta-lactamase gene(VIIIa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 75: 3727-3731(1978)), as well as the tac promoter (DeBoer et al., Proc. Natl. Acad.Sci. USA 80: 21-25 (1993)). Further promoters include trp promoter,phage lambda P_(L), T7 promoter and the like. Promoters suitable for usein the invention are described in Gilbert et al., 1980, “Useful proteinsfrom recombinant bacteria” Sci Am 242:74-94, and Sambrook et al., supra.

For filamentous fungal host cells, suitable promoters include, but arenot limited to, promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase).

The promoter can be any of the promoters listed in U.S. patentapplication Ser. No. 13/330,324. In particular, the promoter can be apromoter region from a portion of the Y. lipolytica gene YALI0E12683, apromoter region from a portion of the Y. lipolytica gene YALI0E19206, ora promoter region from a portion of the Y. lipolytica gene YALI0E34749.In some embodiments, the promoter comprises the nucleotide sequence ofSEQ ID NO:74 (a 0.25 kb sequence of YALI0E12683), SEQ ID NO:75 (a 0.25kb sequence of YALI0E19206), or SEQ ID NO:76 (a 0.25 kb sequence ofYALI0E34749). In some embodiments, the promoter has at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, and at least 99%sequence identity to the nucleotide sequence of SEQ ID NO:74, SEQ IDNO:75, or SEQ ID NO:76.

Other Regulatory Elements

Expression of the exogenous gene may be enhanced by also incorporatingtranscription terminators, leader sequences, polyadenylation sequences,secretory signals, propeptide coding regions, regulatory sequences,and/or selectable markers as would be apparent to one of skill in theart. The choice of appropriate control sequences for use in thepolynucleotide constructs of the present disclosure is within the skillin the art and in various embodiments is dependent on the recombinanthost cell used and the desired method of recovering the fatty alcoholcompositions produced.

Useful regulatory sequences for Yarrowia include, but are not limitedto, Xpr2 promoter fragments (U.S. Pat. No. 6,083,717). Useful terminatorsequences include, but are not limited to, Y. lipolytica Xpr2 (U.S. Pat.No. 4,937,189) and Pox2 (YALI0FI 0857g) terminator sequences.

In various embodiments, the expression vector includes one or moreselectable markers, which permit easy selection of transformed cells.Selectable markers for use in a host organism as described hereininclude, but are not limited to, genes that confers antibioticresistance (e.g., ampicillin, kanamycin, chloramphenicol, hygromycin, ortetracycline resistance) to the recombinant host organism that comprisesthe vector.

VIII. Improved Production of Fatty Acyl-CoA Derivatives

The modified microbial organisms described herein exhibit improvedproduction of fatty acyl-CoA derivatives. The yield of fatty acyl-CoAderivatives of the modified microbial organism of the invention can becompared to a control organism of the same type (e.g., an otherwiseidentical control microbial organism in which the endogenous gene hasnot been disrupted). In one embodiment, the modified microbial organismhas at least one disrupted endogenous gene that is YALI0C17545,YALI0E28336, YALI0E11099, YALI0B10406, YALI0A19536, YALI0E28534,YALI0E32769, YALI0E30283, YALI0E12463, YALI0E17787, YALI0B14014,YALI0A10769, YALI0A15147, YALI0A16379, YALI0A20944, YALI0B07755,YALI0B10175, YALI0B13838, YALI0C02387, YALI0D05511, YALI0D01738,YALI0D02167, YALI0D04246, YALI0D05291, YALI0D07986, YALI0D10417,YALI0D14366, YALI0D25630, YALI0E03212, ALI0E07810, YALI0E12859,YALI0E14322, YALI0E15378, YALI0E15400, YALI0E18502, YALI0E18568,YALI0E22781, YALI0E25982, YALI0E28314, YALI0E32417, YALI0F01320,YALI0F06578, YALI0F07535, YALI0F14729, YALI0F22121, YALI0F25003,YALI0E14792, YALI0B17512, or a homolog of any of these, and an exogenousgene encoding a functional fatty acyl reductase operably linked to apromoter. In some embodiments, the organism exhibits at least a 1.2-foldincrease in the production of fatty acyl-CoA derivatives as compared toa control organism of the same type (e.g., an otherwise identicalcontrol microbial organism in which the one or more endogenous genes arenot disrupted). In other embodiments, the improved production is atleast 1-fold, at least 1.2-fold, at least 1.5-fold, at least 2.5-fold,at least 4-fold, at least 10-fold, at least 15-fold, at least 20-fold,at least 30-fold, at least 40-fold, at least 50-fold, or at least60-fold compared to the control microbial organism. In some embodiments,the exogenous gene encoding a fatty acyl reductase is a gene having atleast 80% sequence identity to the nucleotide sequence of FAR_Maa (SEQID NO:1), FAR_Maq (SEQ ID NO:3), or FAR_Ocs (SEQ ID NO:5). In someembodiments, the exogenous gene encodes a FAR polypeptide having atleast 80% sequence identity to wild-type FAR_Maa (SEQ ID NO:2),wild-type FAR_Maq (SEQ ID NO:4), or wild-type FAR_Ocs (SEQ ID NO:6). Insome embodiments, the exogenous gene encodes a FAR variant derived fromFAR_Maa (SEQ ID NO:2), FAR_Maq (SEQ ID NO:4), or FAR_Ocs (SEQ ID NO:6).

In some embodiments, the invention provides a microbial organism (e.g.,an algae, a bacteria, a mold, a filamentous fungus, a yeast, or anoleaginous yeast) comprising one, two, three, four, or more disruptedendogenous genes wherein at least one of the disrupted endogenous genesis selected from C17545 gene, the E28336 gene, the E11099 gene, theE28534 gene, and homologs thereof, and an exogenous gene encoding afunctional fatty acyl reductase gene operably linked to a promoter,wherein the microbial organism exhibits at least a 1-fold, at least a1.2-fold, at least a 1.5-fold, at least a 2.5-fold, at least a 4-fold,at least a 10-fold, at least a 15-fold, or at least a 20-fold increasein the production of fatty acyl-CoA derivatives as compared to a controlmicrobial organism (e.g., an otherwise identical control microbialorganism in which the one or more genes are not disrupted).

In one embodiment, the invention provides a Yarrowia lipolytica cellcomprising at least one disrupted endogenous gene that is YALI0C17545,YALI0E28336, YALI0E11099, YALI0B10406, YALI0A19536, YALI0E28534,YALI0E32769, YALI0E30283, YALI0E12463, YALI0E14729, or YALI0B17512 or ahomolog of any of these, and an exogenous gene encoding a functionalfatty acyl reductase gene operably linked to a promoter, wherein theYarrowia lipolytica cell exhibits at least a 1-fold increase in theproduction of fatty acyl-CoA derivatives as compared to a controlmicrobial organism (e.g., an otherwise identical control microbialorganism in which the one or more genes are not disrupted). In certainembodiments, the invention provides a Yarrowia lipolytica cellcomprising a disrupted gene or combination of disrupted genes set forthin Table 3 or in Table 4. In certain embodiments, the invention providesa yeast cell comprising a disrupted gene that is a homolog of, orcombination of disrupted genes that are homologs of, the genes set forthin Table 3 or in Table 4.

The control microbial organism can be, e.g., Y. lipolytica DSMZ 1345(wild-type) or Y. lipolytica strain CY-201 (a Y. lipolytica DSMZ 1345variant that grows poorly in growth on media with hexadecane as the solecarbon source). In some embodiments, the control microbial organism is arecombinant organism having the identically incorporated exogenous genesas the microbial organism with the disrupted gene(s). For example, boththe microbial organism having one or more disrupted endogenous genes andthe control microbial organism may contain an exogenous FAR gene.

When comparing the microbial organism having one or more disruptedendogenous genes to the control microbial organism, the organisms shouldbe cultured under essentially identical conditions, and the fattyacyl-CoA derivatives should be measured or recovered using essentiallyidentical procedures.

Fatty Alcohol Production

In some embodiments, the fatty acyl-CoA derivative that is produced is afatty alcohol. Thus, in some embodiments, the invention provides amodified microbial organism that exhibits at least a 1-fold, at least a1.2-fold, at least a 1.5-fold, at least a 2.5-fold, at least a 4-fold,at least a 10-fold, at least a 15-fold, or at least a 20-fold increasein the production of fatty alcohols as compared to a control microbialorganism in which the one or more genes are not disrupted.

Fatty alcohol production can be measured by methods described in theExamples section (e.g., Examples 3 and 6) and/or using any other methodsknown in the art. Fatty alcohol production by an organism of the presentinvention (e.g., a microbial organism having a disrupted endogenousgene) can be described as an absolute quantity (e.g., moles/liter ofculture) or as a fold-improvement over production by a control organism(e.g., a microbial organism in which the endogenous gene was notdisrupted). Fatty alcohol production by a microbial organism of thepresent invention can be measured, for example, using gaschromatography. In general, the microbes are cultured, total or secretedfatty alcohols are isolated, and fatty alcohol amount and/or content ismeasured.

Any number of assays can be used to determine whether a microbialorganism comprising at least one disrupted endogenous gene as describedherein produces an increased amount of fatty alcohols (e.g., at least 1times more fatty alcohols) as compared to a control microbial organismin which the one or more genes are not disrupted, including exemplaryassays described herein. In one exemplary assay, fatty alcohols producedby productive Y. lipolytica strains are collected by extraction of cellcultures using 1 mL of isopropanol:hexane (4:6 ratio). The extractionmixture is centrifuged and the upper organic phase is transferred into a96-well plate and analyzed by gas chromatography (GC) equipped withflame ionization detector (FID) and HP-5 column (length 30 m, I.D. 0.32mm, film 0.25 um), starting at 100° C., and increasing the temperatureat a rate of 25° C./min to 246° C., then holding for 1.96 min.

IX. Methods of Producing Fatty Acyl-CoA Derivatives

The present disclosure also provides methods of producing fatty acyl-CoAderivatives using the microbial organisms as described herein, as wellas the resultant fatty acyl-CoA derivative compositions produced by saidmethods.

Fermentation

Fermentation of the host cell is carried out under suitable conditionsand for a time sufficient to produce fatty acyl-CoA derivatives.Conditions for the culture and production of cells, includingfilamentous fungi, bacterial, and yeast cells, are readily available.Cell culture media in general are set forth in Atlas and Parks, eds.,1993, The Handbook of Microbiological Media. The individual componentsof such media are available from commercial sources, e.g., under theDIFCO™ and BBL™ trademarks. In some embodiments, the aqueous nutrientmedium is a “rich medium” comprising complex sources of nitrogen, salts,and carbon, such as YP medium, comprising 10 g/L of peptone and 10 g/Lyeast extract of such a medium. In other embodiments, the aqueousnutrient medium is Yeast Nitrogen Base (DIFCO™) supplemented with anappropriate mixture of amino acids, e.g., SC medium. In particularembodiments, the amino acid mixture lacks one or more amino acids,thereby imposing selective pressure for maintenance of an expressionvector within the recombinant host cell.

The culture medium can contain an assimilable carbon source. Assimilablecarbon sources are available in many forms and include renewable carbonsources and the cellulosic and starch feedstock substrates obtainedtherefrom. Exemplary assimilable carbon sources include, but are notlimited to, monosaccharides, disaccharides, oligosaccharides, saturatedand unsaturated fatty acids, succinate, acetate and mixtures thereof.Further carbon sources include, without limitation, glucose, galactose,sucrose, xylose, fructose, glycerol, arabinose, mannose, raffinose,lactose, maltose, and mixtures thereof. The culture media can include,e.g., feedstock from a cellulose-containing biomass, a lignocellulosicbiomass, or a sucrose-containing biomass.

In some embodiments, “fermentable sugars” are used as the assimilablecarbon source. “Fermentable sugar” means simple sugars (monosaccharides,disaccharides, and short oligosaccharides) including, but not limitedto, glucose, fructose, xylose, galactose, arabinose, mannose, andsucrose. In one embodiment, fermentation is carried out with a mixtureof glucose and galactose as the assimilable carbon source. In anotherembodiment, fermentation is carried out with glucose alone to accumulatebiomass, after which the glucose is substantially removed and replacedwith an inducer, e.g., galactose for induction of expression of one ormore exogenous genes involved in fatty acyl-CoA derivative production.In still another embodiment, fermentation is carried out with anassimilable carbon source that does not mediate glucose repression,e.g., raffinose, to accumulate biomass, after which the inducer, e.g.,galactose, is added to induce expression of one or more exogenous genesinvolved in fatty acyl-CoA derivative production. In some embodiments,the assimilable carbon source is from cellulosic and starch feedstockderived from but not limited to, wood, wood pulp, paper pulp, grain,corn stover, corn fiber, rice, paper and pulp processing waste, woody orherbaceous plants, fruit or vegetable pulp, distillers grain, grasses,rice hulls, wheat straw, cotton, hemp, flax, sisal, corn cobs, sugarcane bagasse, switch grass, and mixtures thereof.

In one embodiment, the method of making fatty acyl-CoA derivativesfurther includes the steps of contacting a cellulose-containing biomasswith one or more cellulases to yield a feedstock of fermentable sugars,and contacting the fermentable sugars with a microbial organism asdescribed herein. In one embodiment, the microbial organism is Y.lipolytica, and the fermentable sugars are glucose, sucrose, and/orfructose.

The microorganisms can be grown under batch, fed-batch, or continuousfermentations conditions, which are all known in the art. Classicalbatch fermentation is a closed system, wherein the compositions of themedium is set at the beginning of the fermentation and is not subject toartificial alternations during the fermentation. A variation of thebatch system is a fed-batch fermentation, where the substrate is addedin increments as the fermentation progresses. Fed-batch systems areuseful when catabolite repression is likely to inhibit the metabolism ofthe cells and where it is desirable to have limited amounts of substratein the medium. Continuous fermentation is an open system where a definedfermentation medium is added continuously to a bioreactor, and an equalamount of conditioned medium is removed simultaneously for processing.Continuous fermentation generally maintains the cultures at a constanthigh density where cells are primarily in log phase growth. Continuousfermentation systems strive to maintain steady state growth conditions.Methods for modulating nutrients and growth factors for continuousfermentation processes as well as techniques for maximizing the rate ofproduct formation are well known in the art of industrial microbiology.

In some embodiments, fermentations are carried out a temperature ofabout 10° C. to about 60° C., about 15° C. to about 50° C., about 20° C.to about 45° C., about 20° C. to about 40° C., about 20° C. to about 35°C., or about 25° C. to about 45° C. In one embodiment, the fermentationis carried out at a temperature of about 28° C. and/or about 30° C. Itwill be understood that, in certain embodiments where thermostable hostcells are used, fermentations may be carried out at higher temperatures.

In some embodiments, the fermentation is carried out for a time periodof about 8 hours to 240 hours, about 8 hours to about 168 hours, about 8hours to 144 hours, about 16 hours to about 120 hours, or about 24 hoursto about 72 hours.

In some embodiments, the fermentation will be carried out at a pH ofabout 3 to about 8, about 4.5 to about 7.5, about 5 to about 7, or about5.5 to about 6.5.

In one embodiment, the method of producing fatty acyl-CoA derivativescomprises:

-   -   a) providing a microbial organism (e.g., a Yarrowia lipolytica        cell) having one or more disrupted endogenous genes, wherein at        least one disrupted gene is YALI0C17545, YALI0E28336,        YALI0E11099, YALI0B10406, YALI0A19536, YALI0E28534, YALI0E32769,        YALI0E30283, YALI0E12463, YALI0E17787, YALI0B14014, YALI0A10769,        YALI0A15147, YALI0A16379, YALI0A20944, YALI0B07755, YALI0B10175,        YALI0B13838, YALI0D02387, YALI0D05511, YALI0D01738, YALI0D02167,        YALI0D04246, YALI0D05291, YALI0D07986, YALI0D10417, YALI0D14366,        YALI0D25630, YALI0E03212, ALI0E07810, YALI0E12859, YALI0E14322,        YALI0E15378, YALI0E15400, YALI0E18502, YALI0E18568, YALI0E22781,        YALI0E25982, YALI0E28314, YALI0E32417, YALI0F01320, YALI0F06578,        YALI0F07535, YALI0F14729, YALI0F22121, YALI0F25003, YALI0E14720,        YALI0B17512, or a homolog of any of these, and an exogenous gene        encoding a functional fatty acyl reductase operably linked to a        promoter; and    -   b) culturing the microbial organism (e.g., the Yarrowia cell) to        allow production of a fatty acyl-CoA derivative, wherein the        culturing conditions include a temperature of about 20° C. to        about 40° C., a time period of about 16 to about 120 hours, and        a culture medium containing fermentable sugars obtained from a        cellulosic feedstock.

In another embodiment, the above method is modified to include a culturemedium containing sucrose. In some embodiments, wherein the culturemedium contains sucrose, the microbial organism (e.g., the Yarrowiacell) further comprises an exogenous gene encoding an invertase (e.g.,Saccharomyces cerevisiae SUC2 invertase).

In some embodiments, the method of producing fatty acyl-CoA derivativesyields at least 0.5 g/L fatty acyl-CoA derivatives as described below.

Production Levels

The methods described herein produce fatty acyl-CoA derivatives in highyield. Routine culture conditions, e.g., culture of yeast, for such asYarrowia lipolytica, may yield about 0.5 g to about 35 g fatty acyl-CoAderivatives, e.g., fatty alcohols, per liter of culture medium (e.g.,nutrient medium), depending upon the gene(s) disrupted. In someembodiments, the amount of fatty acyl-CoA derivatives, e.g., fattyalcohols, produced by the methods described herein is at least 0.5 g/L,at least 1 g/L, at least 1.5 g/L, at least 2 g/L, at least 2.5 g/L, atleast 3 g/L, at least 3.5 g/L, at least 4 g/L, at least 4.5 g/L, atleast about 5 g/L, or at least 10 g/L, at least 20 g/L, at least 30 g/L,at least 40 g/L, or at least 50 g/L of culture medium.

In some embodiments, the amount of fatty acyl-CoA derivatives, e.g.,fatty alcohols, produced by the methods described herein is about 40mg/g to about 1 g/g, about 40 mg/g to about 5 g/g, about 100 mg/g toabout 1 g/g, about 100 mg/g to about 5 g/g, about 500 mg/g to about 2g/g, about 1 g/g to about 4 g/g, or about 2 g/g to about 3 g/g of drycell weight by routine modification of culturing conditions.

In certain embodiments, the amount of fatty acyl-CoA derivatives, e.g.,fatty alcohols, produced by the methods described herein is about 4% toabout 20%, about 10% to about 20%, about 20% to about 30%, about 30% toabout 40%, about 40% to about 50%, about 50% to about 60%, about 60% toabout 70%, or about 70% to about 80% of dry cell weight by routinemodification of culturing conditions.

Recovery of Fatty Acyl-CoA Derivatives

The methods can further include a step of recovering, e.g., isolating,the fatty acyl-CoA derivatives to yield fatty acyl-CoA derivativecompositions. Recovering or isolating the produced fatty acyl-CoAderivatives refers to separating at least a portion of the fattyacyl-CoA derivatives from other components of the culture medium orfermentation process. Suitable protocols for recovering or isolatingfatty acyl-CoA derivatives from recombinant host cells and/or culturemedium (e.g., distillation, chromatography) are known to the skilledartisan. In certain embodiments, the derivatives are purified (e.g.,substantially free of organic compounds other than the derivative(s)).The derivatives can be purified using purification methods well known inthe art.

In some embodiments, recombinant microorganism hosts secrete the fattyacyl-CoA derivatives into the nutrient medium. In this case, the fattyacyl-CoA derivatives can be isolated by solvent extraction of theaqueous nutrient medium with a suitable water immiscible solvent. Phaseseparation followed by solvent removal provides the fatty acyl-CoAderivative, which may then be further purified and fractionated usingmethods and equipment known in the art. In other embodiments, thesecreted fatty acyl-CoA derivatives coalesce to form a water immisciblephase that can be directly separated from the aqueous nutrient mediumeither during the fermentation or after its completion.

In some embodiments, fatty acyl-CoA derivatives, e.g., fatty alcohols,are isolated by separating the cells from the aqueous nutrient medium,for example by centrifugation, resuspension, and extraction of the fattyacyl-CoA derivatives from the recombinant host cells using an organicsolvent or solvent mixture.

For microorganism hosts that do not secrete the fatty acyl-CoAderivatives into the nutrient media, the fatty acyl-CoA derivatives canbe recovered by first lysing the cells to release the fatty acyl-CoAderivatives and extracting the fatty acyl-CoA derivatives from thelysate using conventional means. See Clontech Laboratories, Inc., 2009,Yeast Protocols Handbook, 100:9156-9161.

X. Fatty acyl-CoA derivatives

As described above, the fatty acyl-CoA derivatives include variouscompounds produced enzymatically by cellular metabolic pathways as shownin FIG. 1. Genetic modification of the enzymes involved in thesepathways can preferentially yield particular derivatives, e.g., fattyalcohols. See Section VII above. Additionally or alternatively,particular fatty acyl-CoA derivatives can be chemically modified (inculture or post-recovery) to yield a different derivative.

The fatty acyl-CoA derivative compositions can include saturated (e.g.,monounsaturated), unsaturated, and branched fatty acyl-CoA derivatives,e.g., fatty alcohols. In some embodiments, the amount of unsaturatedfatty acyl-CoA derivatives (e.g., fatty alcohols) can be less than 50%,less than 40%, less than 30%, less than 20%, less than 10%, less than5%, or less than 1% of the total fatty acyl-CoA derivative composition.In some embodiments, the amount of saturated fatty acyl-CoA derivativescan be less than 50%, less than 40%, less than 30%, less than 20%, lessthan 10%, less than 5%, or less than 1% of the total fatty acyl-CoAderivative composition. In some embodiments, the amount of branchedfatty acyl-CoA derivatives can be less than 50%, less than 40%, lessthan 30%, less than 20%, less than 10%, less than 5%, or less than 1% ofthe total fatty acyl-CoA derivative composition.

In some embodiments, fatty acyl-CoA derivatives (e.g., fatty alcohols,fatty esters, alkanes, alkenes, etc.) having a carbon chain length of C8to C20, C10 to C18, C14 to C18, or C16 to C18 comprise at least 80%, atleast 85%, at least 90%, at least 92%, at least 95%, at least 97%, or atleast 99% by weight of the total fatty acyl-CoA derivative composition.In some embodiments, fatty alcohols having a carbon chain length of C8to C20, C10 to C18, C14 to C18, or C16 to C18 comprise at least 80%, atleast 85%, at least 90%, at least 92%, at least 95%, at least 97%, or atleast 99% by weight of a total fatty alcohol composition. In someembodiments, the fatty acyl-CoA derivatives (e.g., fatty alcohols) havea carbon chain length of C16 to C18. Such C16 to C18 fatty acyl-CoAderivatives, e.g., fatty alcohols, can be saturated, unsaturated, or amixture of saturated and unsaturated derivatives. When the derivative isan alkane or alkene, it is noted that alkanes and/or alkenes havingparticular carbon chain lengths can be isolated from longer and/orshorter alkanes and/or alkenes, for example by HPLC.

In some embodiments, the fatty acyl-CoA derivative is a fatty alcohol.The fatty alcohol can be one or more of 1-octanol (C8:0), 1-decanol(C10:0), 1-dodecanol (C12:0), 1-tetradecanol (C14:0), 1-hexadecanol(C16:0), 1-octadecanol (C18:0), 1-icosanol (C20:0), 1-docosanol,1-tetracosanol, hexadecenol (C16:1), and octadecenol (C18:1).

Alkane and/or Alkene Compositions

In some embodiments, the fatty acyl-CoA derivative is an alkane and/oralkene. The alkanes and/or alkenes can be isolated from the reactionmixture (which may contain unreduced fatty alcohols) to yield acomposition comprising substantially all alkanes and/or alkenes.Alternatively, the alkanes/alkenes and un-reduced fatty alcohols can beisolated from the reaction mixture to yield a composition comprisingalkanes and/or alkenes and fatty alcohols. In some embodiments, thefatty acyl-CoA derivative compositions comprise at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 85%, at least 90%, at least 92%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% alkanesand/or alkenes by weight of the composition after reduction.

In some embodiments, the alkane is octane, decane, dodecane,tetradecane, hexadecane, octadecane, icosane, docosane, tetracosane, ormixtures thereof. In some embodiments, the alkene is octene, decene,dodecene, tetradecene, hexadecene, octadecene, icosene, docosene,tetracosene, or mixtures thereof.

In some embodiments, fatty alcohols produced according to the methodsdescribed herein can be reduced to yield alkanes and/or alkenes havingthe same carbon chain length as the fatty alcohol starting materials.Without being bound by any particular theory, the hydroxyl group of analcohol is a poor leaving group, and therefore, in principle a chemicalmoiety that binds to the oxygen atom of the hydroxyl group to make it abetter leaving group can be used to reduce the fatty alcohols describedherein.

Any method known in the art can be used to reduce the fatty alcohols. Insome embodiments, reduction of fatty alcohols can be carried outchemically, for example, by a Barton deoxygenation (or Barton-McCombiedeoxygenation), a two-step reaction in which the alcohol is firstconverted to a methyl xanthate or thioimidazoyl carbamate, and thexanthate or thioimidazoyl carbamate is reduced with a tin hydride ortrialkylsilane reagent under radical conditions to produce the alkaneand/or alkene. See Li et al., 2007, Modern Organic Synthesis in theLaboratory, p. 81-83.

In another embodiment, alkanes can be produced by hydrogenation of fattyalcohols or fatty acids.

Ester Compositions

In other embodiments, fatty alcohols are reacted with a carboxylic acidto form acid esters. Esterification reactions of fatty alcohols arewell-known in the art. In certain embodiments, the transesterificationreaction is carried out in the presence of a strong catalyst, e.g., astrong alkaline such as sodium hydroxide. In other embodiments, anesterification reaction is carried out enzymatically using an enzymethat catalyzes the conversion of fatty alcohols to acid esters, such aslipoprotein lipase. See, e.g., Tsujita et al., 1999, “Fatty Acid AlcoholEster-Synthesizing Activity of Lipoprotein Lipase” J. Biochem.126:1074-1079.

XI. Exemplary Compositions Comprising Fatty Acyl-CoA Derivatives

In yet another aspect, the present invention relates to the use of themicrobial organisms as described herein for the production of variouscompositions, including but not limited to, fuel compositions (e.g.,biodiesels and petrodiesels), detergent compositions (e.g., laundrydetergents in liquid and powder form, hard surface cleaners, dishwashingliquids, and the like); industrial compositions (e.g., lubricants,solvents; and industrial cleaners); and personal care compositions(e.g., soaps, cosmetics, shampoos, and gels).

Fuel Compositions

In certain embodiments, the fatty acyl-CoA derivative compositionsdescribed herein can be used as components of fuel compositions. Incertain embodiments, the fatty acyl-CoA derivatives produced by themethods described above can be used directly in fuel compositions. Fuelcompositions containing fatty acyl-CoA derivatives produced by themethods of the present invention include any compositions used inpowering combustion engines, including but not limited to biodieselfuels and petrodiesel fuels (e.g., jet fuels and rocket fuels).

In some embodiments, the fuel composition is diesel fuel. Diesel fuel isany fuel used in diesel engines and includes both petrodiesel andbiodiesel. Petrodiesel is a specific fractional distillate of fossilfuel oil. It is comprised of about 75% saturated hydrocarbons and 25%aromatic hydrocarbons. Biodiesel is not derived from petroleum but fromvegetable oil or animal fats and contains long chain alkyl esters.Biodiesel is made by the transesterification of lipids (e.g., spentvegetable oil from fryers or seed oils) with an alcohol and burnscleaner than petrodiesel. Biodiesel can be used alone or mixed withpetrodiesel in any amount for use in modern engines.

In some embodiments, the fuel composition is kerosene. Kerosene is acombustible hydrocarbon that is also a specific fractional distillate offossil fuel and contains hydrocarbons having 6 to 16 carbon atoms.Kerosene has a heat of combustion comparable to that of petrodiesel andis widely used in jet fuel to power jet engines and for heating incertain countries. Kerosene-based fuels can also be burned with liquidoxygen and used as rocket fuel (e.g., RP-1).

In particular embodiments, fatty esters are used as components ofbiodiesel fuel compositions. In various embodiments, fatty acid estersare used as biodiesel fuel without being mixed with other components. Incertain embodiments, the fatty acid esters are mixed with othercomponents, such as petrodiesel fuel. In other embodiments, alkanesand/or alkenes (e.g., C10 to C14) are used as components of jet fuelcompositions. In other embodiments, alkanes and/or alkenes are used ascomponents of rocket fuel. In still other embodiments, alkanes and/oralkenes (e.g., C16 to C24) are used as components in petrodiesel-likefuel compositions.

In some embodiments, the fuel compositions comprise an alkane and/oralkene. In certain embodiments, the alkanes and/or alkenes have from 6to 16 carbons, and the fuel composition is a kerosene-like fuelcomposition. In various embodiments, the kerosene-like fuel compositionsare included in jet fuel compositions. In particular embodiments, thekerosene-like fuel compositions are included in various grades of jetfuel including, but not limited to, grades Avtur, Jet A, Jet A-1, Jet B,JP-4, JP-5, JP-7 and JP-8. In other embodiments, the kerosene-like fuelcompositions are included in fuel compositions for heating. In stillother embodiments, the kerosene-like fuel compositions are burned withliquid oxygen to provide rocket fuel. In particular embodiments, thekerosene-like fuel compositions are used in RP-1 rocket fuel.

In some embodiments, the alkanes and/or alkenes are used in fuelcompositions that are similar to petrodiesel fuel compositions, e.g.,fuels that contain saturated and aromatic hydrocarbons. In certainembodiments, the fuel compositions comprise only alkanes and/or alkenes.In other embodiments, the fuel compositions comprise alkanes and/oralkenes mixed with other components, such as petrodiesel fuel.

In certain embodiments, fatty alcohols, fatty esters, alkanes, and/oralkenes are combined with other fuels or fuel additives to producecompositions having desired properties for their intended use. Exemplaryfuels and fuel additives for particular applications are well-known inthe art. Exemplary fuels that can be combined with the compositionsdescribed herein include, but are not limited to, traditional fuels suchas ethanol and petroleum-based fuels. Exemplary fuel additives that canbe combined with the compositions described herein include, but are notlimited to, cloud point lowering additives, surfactants, antioxidants,metal deactivators, corrosion inhibitors, anti-icing additives,anti-wear additives, deposit-modifying additives, and octane enhancers.

Detergent Compositions

In some embodiments, the fatty acyl-CoA derivative compositionsdescribed herein, and compounds derived therefrom, can be used ascomponents of detergent compositions. Detergent compositions containingfatty acyl-CoA derivatives produced by the methods of the presentinvention include compositions used in cleaning applications, including,but not limited to, laundry detergents, hand-washing agents, dishwashingdetergents, rinse-aid detergents, household detergents, and householdcleaners, in liquid, gel, granular, powder, or tablet form. In someembodiments, the fatty acyl-CoA derivatives (e.g., fatty alcohols)produced by the methods described above can be used directly indetergent compositions. In some embodiments, the fatty acyl-CoAderivatives (e.g., fatty alcohols) can be reacted with a sulfonic acidgroup to produce sulfate derivatives that can be used as components ofdetergent compositions. Detergent compositions that can be generatedusing the fatty acyl-CoA derivatives produced by the methods of thepresent invention include, but are not limited to, hair shampoos andconditioners, carpet shampoos, light-duty household cleaners, light-dutyhousehold detergents, heavy-duty household cleaners, and heavy-dutyhousehold detergents. Detergent compositions generally include, inaddition to fatty acyl-CoA derivatives, one or more or of builders(e.g., sodium carbonate, complexation agents, soap, and zeolites),enzymes (e.g., a protease, a lipase and an amylases); carboxymethylcellulose, optical brighteners, fabric softeners, colourants andperfumes (e.g., cyclohexyl salicylate).

In some embodiments, sulfate derivatives (e.g., C12-15) derived fromfatty acyl-CoA derivatives are used in products such as hair shampoos,carpet shampoos, light-duty household cleaners, and light-duty householddetergents. In some embodiments, sulfate derivatives (e.g., C16-C18)derived from fatty acyl-CoA derivatives are used in products such ashair shampoos and conditioners. In some embodiments, sulfate derivatives(e.g., C16-18) derived from fatty acyl-CoA derivatives are used inproducts such as heavy-duty household cleaners and heavy-duty householddetergents.

Personal Care Compositions

In some embodiments, fatty acyl-CoA derivative compositions as describedherein, and compounds derived therefrom, can be used as components ofpersonal care compositions. In some embodiments, the fatty acyl-CoAderivatives produced by the methods described above can be used directlyin personal care compositions. Personal care compositions containingfatty acyl-CoA derivatives produced by the methods of the presentinvention include compositions used for application to the body (e.g.,for application to the skin, hair, nails, or oral cavity) for thepurposes of grooming, cleaning, beautifying, or caring for the body,including but not limited to lotions, balms, creams, gels, serums,cleansers, toners, masks, sunscreens, soaps, shampoos, conditioners,body washes, styling aids, and cosmetic compositions (e.g., makeup inliquid, cream, solid, anhydrous, or pencil form). In some embodiments,the fatty acyl-CoA derivatives (e.g., fatty alcohols) can be reactedwith a sulfonic acid group to produce sulfate derivatives that can beused as components of said compositions.

In some embodiments, fatty acyl-CoA derivative compositions (e.g., C12)produced by the methods described herein are used in products such aslubricating oils, pharmaceuticals, and as an emollient in cosmetics. Insome embodiments, fatty acyl-CoA derivative compositions (e.g., C14)produced by the methods described herein are used in products such ascosmetics (e.g., cold creams) for its emollient properties. In someembodiments, fatty acyl-CoA derivative compositions (e.g., C16) producedby the methods described herein are used in products such as cosmetics(e.g., skin creams and lotions) as an emollient, emulsifier, orthickening agent. In some embodiments, fatty acyl-CoA derivativecompositions (e.g., C18) produced by the methods described herein areused in products such as lubricants, resins, perfumes, and cosmetics,e.g., as an emollient, emulsifier, or thickening agent. In someembodiments, sulfate derivatives (e.g., C12-14) derived from the fattyacyl-CoA derivative compositions produced by the methods describedherein are used in products such as toothpastes.

Other Compositions

In some embodiments, fatty acyl-CoA derivatives (e.g., fatty alcohols,especially cetyl alcohol, stearyl alcohol and myristyl alcohol) may beused as food additives (e.g., adjuvants and production aids).

XII. EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Expression of Wild-Type M. algicola DG893 FAR in Y. lipolyticaStrains

Wild-type FAR from M. algicola (FAR_Maa) was expressed in Y. lipolyticastrains. The sequence of the codon optimized M. algicola DG893 FAR genecorresponds to SEQ ID NO:1, and the corresponding polypeptide sequenceis designated SEQ ID NO:2. An autonomous replicating plasmid, pCEN354,was constructed for expression of the M. algicola DG893 FAR gene in Y.lipolytica strains. The replicating plasmid was engineered with twoantibiotic selection marker cassettes for resistance to hygromycin andphleomycin (HygB(R) or Ble(R), respectively). Expression of eachcassette is independently regulated by a strong, constitutive promoterisolated from Y. lipolytica: pTEF1 for Ble(R) expression and pRPS7 forHygB(R) expression. Plasmid pCEN354 was used to assemble Y. lipolyticaexpression plasmids. Using “restriction free cloning” methodology, theBle(R) gene was replaced with the M. algicola DG893 FAR gene to providethe plasmid pCEN411 (FIG. 2). In pCEN411, FAR gene expression is undercontrol of the constitutive TEF1 promoter, and the HygB(R) gene allowsfor selection in media containing hygromycin. Ars18 is an autonomousreplicating sequence isolated from Y. lipolytica genomic DNA. Theresulting plasmid was transformed into Y. lipolytica strains usingroutine transformation methods. See, e.g., Chen et al., 1997, “One-steptransformation of the dimorphic yeast Yarrowia lipolytica” ApplMicrobiol Biotechnol 48:232-235.

FAR was also expressed by integrating an expression cassette to aspecific location on the Y. lipolytica genome. In this case, the DNA tobe integrated contained a M. algicola FAR expression cassette and asecond expression cassette that encoded hygromycin resistance. The DNAencoding these expression cassettes was flanked on either side by ˜1 kbof Y. lipolytica DNA that acted to target this DNA to a specificintergenic site on chromosome E. This site was identified as anexpression “hot-spot” by random integration of a FAR expression cassettefollowed by mapping of the integration sites of the most activetransformants. Integration constructs were amplified by PCR andtransformed into Y. lipolytica using routine transformation methods.

Through random integration of a M. algicola FAR expression cassette intothe Y. lipolytica genome, a number of strains were identified withimproved fatty alcohol titers relative to strains with plasmid-based FARexpression. The integration locations in five of the best randomintegrant strains were determined using the “vectorette” PCR method. Ineach of these strains, there were two copies of the FAR gene in either adirect or inverted repeat structure.

One copy of a M. algicola FAR expression cassette was introduced bytargeted integration to either the plus or minus strand of one of fivehot spots identified in the genome of the Y. lipolytica CY-201 strain.Integration site tFARi-1 was located on chromosome E between by 1433493and by 1433495 on the minus strand. Integration site tFARi-2 was locatedon chromosome C between by 2526105 and by 2526114 on the plus strand.Integration site tFARi-3 was located on chromosome B between by 2431420and by 2431425 on the plus strand. Integration site tFARi-4 was locatedon chromosome D between by 1669582 and by 1669588 on the plus strand.Integration site tFARi-5 was located on chromosome D between by 518746and by 518789 on the plus strand. Integration site tFARi-6 was locatedon chromosome B between by 2431420 and by 231425 on the minus strand.Integration site tFARi-7 was located on chromosome D between by 1669582and by 1669588 on the minus strand. Integration site tFARi-8 was locatedon chromosome D between by 518746 and by 518789 on the minus strand.

Example 2 In Vivo Activity of Exogenous M. algicola FAR in RecombinantY. lipolytica Strains

Two Y. lipolytica strains were used for constructing gene knockouts: 1)Y. lipolytica DSMZ 1345 obtained from the German Resource Centre forBiological Material (DSMZ), and 2) Y. lipolytica CY-201, an improvedproduction host obtained by UV-mutagenesis of Y. lipolytica DSMZ 1345and defective in growth on media with hexadecane as the sole carbonsource. When transformed with pCEN411, Y. lipolytica CY-201 produced 7-to 10-fold more fatty alcohols compared to Y. lipolytica DSMZ 1345 andalso significantly reduced the rate of degradation of exogenous1-hexadecanol in YPD media containing 8% glucose and 500 μg/mLhygromycin. The expression of alternative FAR genes and variants inmodified Y. lipolytica strains can be assessed using similarmethodology.

Example 3 Analysis of Fatty Alcohol Production in Y. lipolytica StrainsContaining Exogenous FAR

Y. lipolytica strains comprising a plasmid containing an exogenous geneencoding M. algicola DG893 FAR were grown in 96-well Axygen platescontaining 250 μL YPD supplemented with 2% glucose and 500 μg/mLhygromycin. Plates were incubated in a Kuhner shaker-incubator forapproximately 40-48 hours at 30° C., 200 rpm and 85% relative humidity.The cell cultures were diluted by transferring 50 μL of overnight growncultures into the Axygen 96-well plates containing 250 μL YPDsupplemented with 2% glucose and 500 μg/mL hygromycin. The plates wereincubated for approximately 24-28 hours in a Kuhner shaker-incubatorunder the same conditions. 20 μL of the cell cultures were transferredinto deep 96-well plates containing 380 μL YPD supplemented with 8%glucose and 500 μg/mL hygromycin. The plates were incubated forapproximately 22-26 hours under the same conditions. Cells werecollected by centrifugation for 10 minutes at 3500 rpm. Cell pelletswere resuspended in 400 μL of nitrogen limitation media (1.7 g/L yeastnitrogen base, 1.4 g/L (NH₄)₂SO₄, 30 g/L glucose) containing 500 μg/mLhygromycin and incubated for 22-26 hours in a Kuhner shaker-incubator at30° C., 200 rpm and 85% relative humidity. The cell cultures wereextracted with 1 mL of isopropanol:hexane (4:6 ratio) for 2 hrs. Theextracts were centrifuged, and the upper organic phase was transferredinto polypropylene 96-well plates. Samples were analyzed using thefollowing GC-FID method.

A 1 μL sample was analyzed by GC-FID with a split ratio 1:10 using thefollowing conditions: GC-6890N from Agilent Technologies equipped withFID detection and HP-5 column (length 30 m, I.D. 0.32 mm, film 0.25 um).GC method: start at 100° C., increase the temperature with a rate of 25°C./min to 246° C. and hold for 1.96 min. Total run time, 7.8 min. Underthe above GC conditions, the approximate retention times (min) ofproduced fatty alcohols and acids are as follows: 5.74, C16:1-OH; 5.93,C16:0-OH; 6.11, C16:0-OOMe (internal standard); 6.16, C16:1-OOH; 6.29,C16:0-OOH; 6.80, C18:1-OH; 6.90, C18:0-OH; 7.3, C18:0- and C18:1-OOH.Identification of individual fatty alcohols was done by comparison tocommercial standards (Sigma Chemical Company). Under the conditionstested, expression of the M. algicola DG893 FAR in the parent Y.lipolytica DSMZ 1345 and CY-201 strains resulted in 5-20 mg/L and100-200 mg/L production of fatty alcohols, respectively. The fattyalcohols were produced were: 70-80% C16:0 (1-hexadecanol), 10-15% 18:0(1-octadecanol) and 10-15% C18:1 (cis Δ⁹-1-octadecenol).

Example 4 Identification of Y. lipolytica Gene Targets for Disruption

Genes selected for disruption were identified in several ways. Somegenes were selected based on their roles in pathways for hydrocarbonassimilation in alkane-utilizing yeast. Because fatty acyl-CoA is anintermediate in these pathways resulting from oxidation of alkanes, thestability of fatty acyl-CoA derivatives may be improved by disruptinggenes responsible for alkane utilization. Other genes for disruptionwere selected based on their homology to such genes. These includedgenes whose sequence predicted that they may function as alcoholdehydrogenases or acyltransferases involved in lipid biosynthesis.

Additional genes for disruption include those that encode for proteinsinvolved in import of newly synthesized proteins into the endoplasmicreticulum. These include the subunits of the trimeric protein conductingchannel (Sec61, Ssh1, Sbh1, and Sss1), the tetrameric Sec62/Sec63complex (Sec62, Sec63, Sec66, and Sec72), and other resident endoplasmicreticulum proteins (Kar2 and Sls1) (Boisrame A. et al., “Interaction ofKar2p and Sls1p Is Required for Efficient Co-translational Translocationof Secreted Proteins in the Yeast Yarrowia lipolytica,” J. Biol. Chem.(1998) 273: 30903).

Other genes for disruption were identified by comparison of global geneexpression in glucose and glycerol-based media. In particular, geneswhose expression is repressed by glycerol were selected, since alkaneutilization is repressed in glycerol containing media.Glycerol-repressed genes were identified by microarray analysis usingRNA prepared from Y. lipolytica DSMZ 1345 cultured in both rich mediaand lipid accumulation media.

Example 5 Construction and Analysis of Strains Having Disrupted Genes

Knockout strains can be constructed by transforming Y. lipolytica with aDNA construct designed to replace most or all of the open reading frameof interest with a selectable marker by homologous recombination. Assuch, the DNA constructs may comprise a selectable marker flanked by ˜1kb sequences immediately upstream and downstream of the gene of interestthat are necessary for homologous recombination to occur. These DNAconstructs are contained in plasmids assembled using standard methodsfor plasmid construction. For transformation, the DNA construct ofinterest is amplified from the corresponding plasmid using PCR togenerate ˜1 μg of linear DNA. This DNA is transformed into Y. lipolyticausing the method described in Madzak et al., 2003, “Yarrowialipolytica.” In Gellissen, ed. Production of Recombinant Proteins NovelMicrobial and Eukaryotic Expression Systems, p. 163-189. Strains inwhich the gene of interest is replaced with the selectable marker areidentified by ability to grow on selective media and by PCR genotyping.Typical selective markers are familiar to those skilled in the art (see,e.g., Fickers et al., 2003, “New disruption cassettes for rapid genedisruption and marker rescue in the yeast Yarrowia lipolytica” Journalof Microbiological Methods 55:727-737).

In a second step, the selectable marker is excised from the chromosomeusing methods that are familiar to those skilled in the art. See, e.g.,Fickers et al., supra; Akada et al., 2006, “PCR-mediated seamless genedeletion and marker recycling in Saccharomyces cerevisiae” Yeast23:399-405; Fonzi et al., 1993, “Isogenic strain construction and genemapping in Candida albicans” Genetics 134:717-728. Strains with excisedmarkers can be easily identified by growth on counter-selection media ifthe selectable marker used is bifunctional, i.e., it encodes an enzymewhose product(s) are essential for growth on positive selection mediaand toxic on another selection media. Such bifunctional markers arefamiliar to those skilled in the art.

For construction of strains with multiple gene disruptions, Y.lipolytica can be transformed sequentially with a series of DNAconstructs designed to knockout the genes of interest. Eachtransformation can be carried out by the method described above, suchthat the selectable marker is excised after each disruption step. Thus,any combination of knockouts can be created in a given strain using thecollection of plasmids harboring the DNA constructs described above.

Example 6 Analysis of Fatty Alcohol Production in Modified Y. LipolyticaStrains

A collection of ˜233 strains comprising strains with single genedisruptions and strains with 2 or more gene disruptions were created inboth the DSMZ1345 and CY-201 strain backgrounds. These strains weretransformed with plasmid pCEN411 for expression of wild-type M. algicolaDG893 FAR (see FIG. 2) and screened for fatty alcohol production asdescribed above. Tables 3 and 4 below provide the relative fatty alcoholproduction for targeted gene disruption Y. lipolytica DSMZ 1345 andCY-201 strains expressing wild-type M. algicola DG893 FAR gene relativeto the corresponding Y. lipolytica strain with no targeted gene deletionand expressing wild-type M. algicola DG893 FAR gene. The fatty alcoholsproduced were: 70-80% C16:0 (1-hexadecanol), 10-15% 18:0(1-octadecanol), and 10-15% C18:1 (cis Δ9-1-octadecenol).

TABLE 3 Targeted gene disruptions in Y. lipolytica DSMZ 1345 Fattyalcohol production Gene(s) Disrupted relative to DSMZ 1345 C17545 E28336E11099 E28534 +++++ C17545 E28336 E12463 E28534 +++++ C17545 E28336A19536 E28534 +++++ E28336 E32769 C17545 E28534 +++++ C17545 E28534+++++ C17545 E28336 E12463 +++++ C17545 E28336 E28534 +++++ C17545E28336 A19536 B10406 +++++ E28336 E32769 C17545 B10406 +++++ C17545E28336 E11099 B10406 +++++ C17545 B10406 E11099 +++++ C17545 E28336B10406 +++++ E28336 E32769 C17545 E11099 +++++ C17545 E28336 E12463+++++ C17545 E28336 E11099 +++++ C17545 E28336 A19536 E11099 +++++C17545 E28534 B17512 +++++ E11099 A19536 B10406 B17512 +++++ E28336C17545 ++++ E28336 C17545 ++++ C17545 E28336 E11099 ++++ C17545 E28336A19536 ++++ E11099 A19536 B10406 ++++ E28336 E32769 C17545 ++++ C17545E28336 A19536 ++++ E28336 E32769 C17545 ++++ C17545 E28336 ++++ E28336B10406 E11099 ++++ E11099 E30283 ++++ E28336 E32769 E11099 B10406 ++++C17545 B10406 E11099 ++++ E28336 E30283 ++++ E30283 ++++ C17545 E30283++++ A19536 E30283 ++++ C17545 B10406 A19536 ++++ E12463 E30283 ++++C17545 B10406 ++++ A19536 E28534 ++++ B17512 ++++ E14729 +++ C17545E28336 E17787 +++ E28336 E32769 E28534 +++ E28336 B10406 +++ E28336E28534 +++ E28336 E15378 E12463 +++ C17545 B10406 +++ D25630 A16379E17787 A15147 C17545 +++ C17545 E11099 +++ E28336 E15378 +++ C17545A19536 +++ E28336 E15378 E11099 +++ B10406 E28534 +++ E28336 E15378A19536 +++ E28336 E32769 A19536 B10406 +++ E11099 A19536 C17545 +++E17787 E28534 +++ C17545 A12859 +++ A15147 E17787 A16379 D25630 +++D25630 A16379 E17787 A15147 E11099 +++ C17545 D01738 +++ E28336 E32769A19536 E11099 +++ D25630 A16379 E17787 A15147 A19536 +++ D25630 A16379E17787 A15147 E12463 +++ D25630 A16379 E17787 A15147 E28336 +++ C17545E12463 +++ E28336 E32769 A19536 +++ E28534 +++ A19536 E28336 +++ E28336D01738 +++ E28336 E11099 +++ E28336 A19536 A16379 +++ C17545 +++ E17787C17545 +++ E28336 E32769 +++ E28336 A12859 +++ E28336 B14014 E15378 +++E28336 E32769 E11099 +++ E28336 E32769 A19536 +++ E28336 +++ E28336E32769 +++ E28336 E32769 E11099 +++ E12463 +++ E28336 C02387 +++ A19536+++ E28336 B10406 +++ E17787 E28336 +++ E28336 E32769 E12463 +++ E28336A16379 +++ E28336 E32769 E17787 +++ E28336 E15378 E17787 +++ B10406E11099 +++ E28336 B10175 A16379 +++ E12463 E28336 +++ E11099 A19536E28336 +++ E28336 +++ B10406 E17787 +++ E28336 C02387 B10175 +++ B14014C17545 +++ B10175 B10406 +++ E17787 +++ E11099 +++ B10406 +++ E28336A16379 E15400 +++ E28336 E06831 +++ E15400 +++ A19536 E11099 +++ B14014B10406 +++ E28336 E32769 B10175 +++ B14014 +++ B10175 +++ E11099 B14014+++ D01738 +++ E12859 +++ B13838 +++ D02167 +++ F01320 +++ E28336 E32769B14014 +++ A19536 E15400 +++ B10406 A19536 ++ B07755 ++ B10175 A19536 ++D05291 ++ A15147 E17787 A16379 D25630 ++ E15378 ++ F25003 ++ F22121 ++B14014 A12859 ++ E18502 ++ C02387 ++ C08415 ++ A10769 ++ F06578 ++F07535 ++ D14366 ++ F14729 ++ D07986 ++ E32769 ++ A16379 D25630 ++E03212 ++ D10417 ++ B14014 A16379 ++ E07810 ++ B14014 D25630 ++ E25982++ A20944 ++ E18568 ++ E32417 ++ E28314 ++ E22781 ++ A15147 ++ D04246 ++C05511 ++ E14322 ++ A17875 E15400 B01298 ++ D25322 ++ B04906 ++ F29623++ A06655 ++ E17787 A16379 D25630 ++ F19514 ++ B13816 ++ B00462 ++E12463 A19536 ++ E06567 ++ D17314 ++ B14014 A15147 E17787 A16379 D25630++ E12463 E11099 ++ C23859 ++ E12463 B10406 ++ B08404 ++ D25960 ++A17875 ++ E01210 ++ E27654 ++ A17875 E15400 B01298 F23793 ++ E21560 ++D12628 ++ E06831 ++ D25630 ++ B10175 E17787 + E15818 + E14509 + C19580 +B12386 + A10769 + A16379 B14014 D25630 + B10175 E12463 + B14014 E17787 +F19580 + A17875 B01298 + E01298 + E32835 + D04884 + E17787 E30283 +E28336 C02387 B14014 + F10857 + A07733 + E15400 B10175 + C19096 +D07942 + E15400 B01298 F23793 + E11099 B10175 + E19921 + E12463 E17787 +D00176 + E17787 A19536 + E04961 + A19536 E11099 + C10054 + E15400B01298 + E07766 + C14784 + E18700 + B01298 + E17787 E11099 + F23793 +F23793 B01298 + E12463 A12859 + B21692 + C09284 + D17864 + A00374 +B14014 D01738 + B14014 E12463 + E28336 E15378 B10406 + A16379 + E12419 +E16016 + E12463 D01738 + D27302 + C04092 + E29161 + B05456 + F00748 +D00891 + F22539 + +++++ = >30.0 fold improvement ++++ = 10.0 to 30.0fold improvement +++ = 1.5 to 9.99 fold improvement ++ = 1.0 to 1.49fold improvement + = 0.0 to 0.99 fold improvement

TABLE 4 Targeted gene disruptions in Y. lipolytica CY-201 Relative fattyalcohol Gene(s) Disrupted production to CY-201 E14729 +++++ E11099E28336 C17545 E14729 +++++ C17545 B10406 E28336 ++++ E11099 E28336C17545 A00374 ++++ E11099 A19536 C17545 A00374 ++++ E11099 A19536 C17545++++ C17545 B10406 A19536 ++++ B10406 E14729 ++++ C17545 E14729 ++++E11099 E14729 ++++ E28336 E14729 +++ E11099 E28336 C17545 +++ E11099A19536 C17545 B10406 +++ E11099 E28336 E18502 +++ C17545 B10406 +++E28336 A19536 B10406 +++ E28336 A19536 B10406 +++ E28336 A19536 B10406C17545 +++ C17545 B10406 +++ E28336 E11099 +++ E11099 A19536 C17545 +++C17545 B10406 +++ E28336 A19536 B10406 E11099 +++ E28336 A19536 +++E28336 C17545 +++ E28534 +++ E11099 E28336 E18502 +++ E11099 E28336F09603 +++ C17545 A19536 +++ E28336 +++ E11099 +++ E11099 A19536 B10406+++ C17545 +++ E29336 C08415 +++ E11099 A19536 A00374 +++ A19536 E11099+++ B10406 +++ E11099 A19536 C08415 +++ E28336 A19536 +++ E28336 +++C17545 F09603 ++ C17545 E11099 ++ E12463 E11099 ++ A19536 F09603 ++E12859 ++ E11099 E28336 C17545 A19536 ++ E11099 A19536 ++ A19536 ++E17787 E11099 ++ E11099 A19536 F09603 ++ D02167 ++ E28336 F09603 ++E28336 E32769 ++ B13838 ++ A00374 ++ E18502 ++ E11099 A19536 E18502 ++F09603 ++ C08415 ++ A20944 ++ F01320 ++ D04246 ++ F14729 ++ C04092 ++E11099 F09603 ++ F19514 ++ D07942 ++ E11099 E18502 ++ F07535 ++ B07755++ B21692 ++ B05456 ++ C19580 ++ F22121 ++ E11099 C08415 ++ D05291 ++D01738 ++ F25003 ++ E11099 E28336 B10406 ++ E03212 + D10417 + B10175 +E25982 + A16379 D25630 + A19536 C08415 + B10406 F09603 + D25630 +A17875 + E28336 D07986 E32769 + B04906 + D14366 + E32769 + E17787 +B10406 E28336 + F10857 + F19514 E32769 + C02387 + D07986 E32769 + B14014D25630 + D25630 A16379 E17787 + F06578 E32769 + E28336 C02387 + F06578 +B14014 D25630 E17787 + F19514 F06578 D07986 E32769 + E17787 A16379D25630 B14014 + B14014 + A15147 E17787 B14014 A16379 D25630 + F19514F06578 E32769 + A16379 B14014 D25630 + B10406 E11099 + F06578 D07986E32769 + E32417 + E01298 + E06831 + E16016 + E28336 E15378 + E30283 +B10406 E18502 + F19514 D07986 E32769 + E15378 + E28336 A00374 + E15400F23793 + D00891 + E15400g + B01298 F23793 + A16379 D25630 A15147 +E12463 + A17875 F23793 + B10406 A19536 + F22539 + F23793 + E28336 A19536A00374 + E28336 A19536 C17545 + ++++ = >4.0 fold improvement +++ = 1.5to 4.0 fold improvement ++ = 1.0 to 1.49 fold improvement + = 0.0 to0.99 fold improvement

Example 7 Production of Fatty Alcohol in Fermentation with a Modified Y.Lipolytica Strain

A derivative of the CY-201 strain comprising deletions of YALI0E11099g,YALI0E28336g, YALI0C17545, and YALI0E14729 and harboring two integratedcopies of M. algicola FAR (“the CY-202 strain”) was used to producefatty alcohol in a stirred tank fermentor. The fermentation followed atwo-stage protocol in which cells are propagated in a nutrient-richmedium then transferred into a nutrient limited medium for fatty alcoholproduction. For the first stage, an inoculation culture was prepared bygrowing the CY-202 strain in YPD medium (10 g/L yeast extract, 20 g/Lpeptone, 20 g/L dextrose) in a baffled shake flask at 30° C. for 24hours. This culture was used to inoculate a fermentor containing 10 Lpropagation medium (6.7 g/L Yeast Nitrogen base without amino acids,20.9 g/L Bis Tris buffer, 80 g/L glucose, 10 g/L corn steep liquor, and0.22 mL/L antifoam (a 1:1 mixture of poly(propylene glycol) and AntifoamB), adjusted to pH 6.5 with KOH. This propagation culture was grown at30° C. in a batch process with controlled oxygen transfer rate (15-20 mMO₂/hr) to a final OD₆₀₀ of 12-18. For the second stage, cells inpropagation medium were harvested by centrifugation, then resuspended in1.1 L fatty alcohol production medium (200 g/L glucose, 1 g/L KH₂PO₄, 5g/L (NH₄)₂SO₄. 2.5 mg/L MgSO₄*7H₂0 1 mg/L FeSO₄*7H₂0, 0.5 mg/L H₃BO₃,0.5 mg/L MnSO₄—H₂0, 0.5 mg/L Na₂MoO₄-2H₂0, 0.5 mg/L ZnSO₄*7H₂0, 0.5 mg/LCoCl₂*H₂0, 0.1 mg/L KI, 0.1 mg/L CuCl₂*2H₂0, 50 mg/L Thiamine HCl, and50 mg/L inositol, and 0.8 mL/L antifoam). The volume of the cellresuspension was adjusted to give an initial cell density for the secondstage of 20 g/L (dry cell weight), then the resuspension was loaded intoa stirred tank fermentor. Fermentation was carried out in a batchprocess at 30° C. with dissolved oxygen control (30% dO₂). pH wascontrolled at 3.5 by addition of KOH. Glucose was added as necessary toprevent glucose exhaustion (35 g/L over the course of the fermentation).

Samples were collected at 24 hrs, 48 hrs, and 72 hrs after inoculationof the production stage culture. Fatty alcohol titer was analyzed byGC-FID essentially as described in Example 3. After 24 hrs, a fattyalcohol titer of 9 g/L was observed. After 48 hrs, a fatty alcohol titerof 16 g/L was observed. After 72 hrs, a fatty alcohol titer of 21 g/Lwas observed.

Example 8 Partial Deletion of Sec62 Gene

Strains with a partial deletion of the Sec62 gene (YALI0B17512g; SEQ IDNO:54) were constructed by transforming Y. lipolytica with a DNAconstruct designed to (1) mutate codon Trp235 to a stop codon and (2)replace codons 236-396 with a selectable marker by homologousrecombination. Thirty nucleotides of the 3′ untranslated regionimmediately following the Sec62 coding sequence were also deleted. Thispartial deletion corresponds to a deletion of the cytoplasmic domain ofSec62, which begins immediately after the predicted transmembrane domainat Leu206 and continues to the end of the protein at Glu396. As shown inTable 3, this strain (identified in the table as “B17512”) gave ˜10-foldgreater fatty alcohol production relative to the corresponding DSMZ 1345strain without a partial deletion of the Sec62 gene.

Three other partial deletions of the Sec62 gene (YALI0B17512g) were madeby transforming Y. lipolytica with a DNA construct that (1) mutatedeither the codon encoding Glu267, the codon encoding Ala302, or thecodon encoding Ile337 to a stop codon and (2) replaced the subsequentcodons with a selectable marker using homologous recombination. Thesestrains gave ˜1.5- to 2-fold greater fatty alcohol production relativeto the corresponding DSMZ 1345 strain without a partial deletion of theSec62 gene.

The methods used for DNA construction and transformation are describedin Example 4 and are familiar to those skilled in the art. Briefly, thetransforming DNA construct comprised a bifunctional selectable markerflanked by ˜1 kb of genomic sequences immediately upstream anddownstream of the nucleotides to be deleted. Following transformation,strains with the desired modification were selected by growth onpositive selective media. The selectable marker was then excised fromthe genome, and strains that had lost the marker were identified bygrowth on counterselection media. PCR genotyping was used to confirmthat strains have the desired modification.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A Y. lipolytica recombinant yeast cellcomprising: (a) at least one genetic modification in the YALI0C17545endogenous gene and at least one genetic modification in the YALI0E28534endogenous gene; and (b) an exogenous gene encoding a fatty acylreductase (FAR) protein, wherein the exogenous gene is operably linkedto a promoter, wherein the FAR protein has at least 80% amino sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, 4, 6, and 65-73, wherein the at least one geneticmodification comprises: (i) a deletion of all or a portion of the codingregion of the endogenous gene; (ii) a mutation in the endogenous genesuch that the gene encodes a polypeptide having reduced activity; (iii)a modified regulatory sequence that reduces expression of the endogenousgene; or (iv) any combination of (i)-(iii).
 2. The Y. lipolyticarecombinant yeast cell of claim 1 further comprising at least onegenetic modification in the YALI0E28336 endogenous gene and/or theYALI0E11099 endogenous gene, wherein the at least one geneticmodification comprises: (i) a deletion of all or a portion of the codingregion of the endogenous gene; (ii) a mutation in the endogenous genesuch that the gene encodes a polypeptide having reduced activity; (iii)a modified regulatory sequence that reduces expression of the endogenousgene; or (iv) any combination of (i)-(iii).
 3. The Y. lipolyticarecombinant yeast cell of claim 2 comprising at least one geneticmodification in each of the YALI0E28336 endogenous gene and theYALI0E11099 endogenous gene, wherein the at least one geneticmodification comprises: (i) a deletion of all or a portion of the codingregion of the endogenous gene; (ii) a mutation in the endogenous genesuch that the gene encodes a polypeptide having reduced activity; (iii)a modified regulatory sequence that reduces expression of the endogenousgene; or (iv) any combination of (i)-(iii).
 4. The Y. lipolyticarecombinant yeast cell of claim 1 further comprising at least onegenetic modification in at least one endogenous gene selected from thegroup consisting of YALI0B10406, YALI0A19536, YALI0E32769, YALI0E30283,YALI0E12463, YALI0E17787, YALI0B14014, YALI0A10769, YALI0A15147,YALI0A16379, YALI0A20944, YALI0B07755, YALI0B10175, YALI0B13838,YALI0C02387, YALI0C05511, YALI0D01738, YALI0D02167, YALI0D04246,YALI0D05291, YALI0D07986, YALI0D10417, YALI0D14366, YALI0D25630,YALI0E03212, YALI0E07810, YALI0E12859, YALI0E14322, YALI0E15378,YALI0E15400, YALI0E18502, YALI0E18568, YALI0E22781, YALI0E25982,YALI0E28314, YALI0E32417, YALI0F01320, YALI0F06578, YALI0F07535,YALI0F14729, YALI0F22121, YALI0F25003, YALI0E14729, and YALI0B17512,wherein the at least one genetic modification comprises: (i) a deletionof all or a portion of the coding region of the endogenous gene; (ii) amutation in the endogenous gene such that the gene encodes a polypeptidehaving reduced activity; (iii) a modified regulatory sequence thatreduces expression of the endogenous gene; or (iv) any combination of(i)-(iii).
 5. The Y. lipolytica recombinant yeast cell of claim 4comprising at least one genetic modification in the YALI0B17512endogenous gene, wherein the at least one genetic modificationcomprises: (i) a deletion of all or a portion of the coding region ofthe endogenous gene; (ii) a mutation in the endogenous gene such thatthe gene encodes a polypeptide having reduced activity; (iii) a modifiedregulatory sequence that reduces expression of the endogenous gene; or(iv) any combination of (i)-(iii).
 6. The Y. lipolytica recombinantyeast cell of claim 1, wherein multiple copies of the exogenous gene areexpressed.
 7. The Y. lipolytica recombinant yeast cell of claim 1,further comprising a second exogenous gene that encodes a fatty acidsynthase (FAS), an ester synthase, an acyl-ACP thioesterase (TE), afatty acyl-CoA synthase (FACS), an acetyl-CoA carboxylase (ACC), axylose isomerase, or an invertase.
 8. The Y. lipolytica recombinantyeast cell of claim 1, wherein said Y. lipolytica recombinant yeast cellexhibits at least one-fold increase in the production of a fattyacyl-CoA derivative relative to the corresponding Y. lipolytica yeastcell lacking the at least one modification in the YALI0C17545 endogenousgene and the at least one genetic modification in the YALI0E28534endogenous gene.
 9. An isolated Y. lipolytica recombinant yeast cellcomprising (i) an exogenous gene encoding a fatty acyl reductase (FAR)protein, wherein the exogenous gene is operably linked to a promoter,wherein the FAR protein has at least 80% amino sequence identity to anamino acid sequence selected from the group consisting of SEQ ID NO: 2,4, 6, and 65-73, and (ii) at least one genetic modification in each ofthe two or more endogenous genes selected from: (a) the YALI0C17545 andYALI0E28534 endogenous genes; (b) the YALI0C17545, YALI0E28336, andYALI0E28534 endogenous genes; (c) the YALI0C17545, YALI0E28534, andYALI0B17512 endogenous genes; and (d) the YALI0C17545, YALI0E28336,YALI0E11099, and YALI0E28534 endogenous genes, wherein the at least onegenetic modification comprises: (A) a deletion of all or a portion ofthe coding region of the endogenous gene; (B) a mutation in theendogenous gene such that the gene encodes a polypeptide having reducedactivity; (C) a modified regulatory sequence that reduces expression ofthe endogenous gene; or (D) any combination of (A)-(C).
 10. A method ofproducing a fatty acyl-CoA derivative, the method comprising culturingthe Y. lipolytica recombinant yeast cell of claim 1 under conditions inwhich the fatty acyl-CoA derivative is produced.
 11. The method of claim10, wherein the fatty acyl-CoA derivative is a fatty alcohol, fattyacid, fatty aldehyde, fatty ester, fatty acetate, wax ester, alkane, oralkene.
 12. The method of claim 10, comprising: contacting acellulose-containing biomass with one or more cellulases to yieldfermentable sugars; and contacting said Y. lipolytica recombinant yeastcell with the fermentable sugars under conditions in which the fattyacyl-CoA derivative is produced.
 13. The method of claim 10, wherein atleast 5 g/L of fatty acyl-CoA derivatives per liter of culture medium isproduced.
 14. An isolated Y. lipolytica recombinant yeast cellcomprising at least one genetic modification in the YALI0C17545endogenous gene and at least one genetic modification in the YALI0E28534endogenous gene; wherein the at least one genetic modificationcomprises: (i) a deletion of all or a portion of the coding region ofthe endogenous gene; (ii) a mutation in the endogenous gene such thatthe gene encodes a polypeptide having reduced activity; (iii) a modifiedregulatory sequence that reduces expression of the endogenous gene; or(iv) any combination of (i)-(iii).